Assays for the detection of microtubule depolymerization inhibitors

ABSTRACT

This invention provides methods for the screening and identification of agents having potent effects on the progression of the cell cycle. In one embodiment, the methods involve contacting a polymerized microtubule with a microtubule severing protein or a microtubule depolymerizing protein in the presence of an ATP or a GTP and a test agent; and (ii) detecting the formation of tubulin monomers, dimers or oligomers. The p60 subunit of katanin provides a particularly preferred microtubule severing protein possessing both ATPase and microtubule severing activities.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. § 119(e) of provisionalpatent U.S. Ser. No. 60/081,734, filed on Apr. 14, 1998, which is hereinincorporated by reference in its entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

[ Not Applicable ]

FIELD OF THE INVENTION

This invention relates assay for agents that modulate (e.g. upregulate,downregulate or completely inhibit) microtubule depolymerizing ormicrotubule severing proteins. Such agents will have profound effects onprogression of the cell cycle and act as potent anti-mitotic agents.

BACKGROUND OF THE INVENTION

The cytoskeleton constitutes a large family of proteins that areinvolved in many critical processes of biology, such as chromosome andcell division, cell motility and intracellular transport (Vale andKreis, (1993) Guidebook to the Cytoskeletal and Motor Proteins New York:Oxford University Press; Alberts et al., (1994) Molecular Biology of theCell, 788-858). Cytoskeletal proteins are found in all cells and areinvolved in the pathogenesis of a large range of clinical diseases. Thecytoskeleton includes a collection of polymer proteins, microtubules,actin, intermediate filaments, and septins, as well as a wide variety ofproteins that bind to these polymers (polymer-interacting proteins).Some of the polymer-interacting proteins are molecular motors (myosins,kinesins, dyneins) (Goldstein (1993) Ann. Rev. Genetics 27: 319-351;Mooseker and Cheney (1995) Annu. Rev. Cell Biol. 11: 633-675) that areessential for transporting material within cells (e.g., chromosomalmovement during metaphase), for muscle contraction, and for cellmigration. Other groups of proteins (e.g., vinculin, talin andalpha-actinin) link different filaments, connect the cytoskeleton to theplasma membrane, control the assembly and disassembly of thecytoskeletal polymers, and moderate the organization of the polymerswithin cells.

Given the central role of the cytoskeleton in cell division, cellmigration, inflammation, and fungal/parasitic life cycles, it is afertile system for drug discovery. Although much is known about themolecular and structural properties of cytoskeletal components,relatively little is known about how to efficiently manipulatecytoskeletal structure and function. Such manipulation requires thediscovery and development of specific compounds that can predictably andsafely alter cytoskeletal structure and function. However, at present,drug targets in the cytoskeleton have been relatively untapped.Extensive work has been directed towards drugs that interact with thecytoskeletal polymers themselves (e.g., taxol and vincristine), andtowards motility assays (Turner et al. (1996) Anal. Biochem. 242 (1):20-5; Gittes et al. (1996) Biophys. J. 70 (1): 418-29; Shirakawa et al.(1995) J. Exp. Biol. 198: 1809-15; Winkelmann et al. (1995) Biophys. J.68: 2444-53; and Winkelmann et al. (1995) Biophys. J. 68: 72S).Virtually no effort has been directed to finding drugs that target thecytoskeletal proteins that bind to the different filaments, which mightbe more specific targets with fewer unwanted side effects.

SUMMARY OF THE INVENTION

This invention pertains to the discovery that proteins (e.g. motorproteins) that either depolymerize or sever microtubules, provide goodtargets for modulators of such activity. Without being bound by aparticular theory, it is believed that microtubule depolymerizing orsevering activity is critical for normal formation and/or function ofthe mitotic spindle. Thus, agents that modulate (e.g., upregulate,downregulate, or completely inhibit) depolymerization or severingactivity are expected to have a significant activity on progression ofthe cell cycle (e.g. acting as potent anti-mitotic agents).

This invention thus provides, in one embodiment, assays for identifyingan agent that modulates microtubule depolymerization. The assays involvecontacting a polymerized microtubule with a microtubule severing proteinor a microtubule depolymerizing protein in the presence of an ATP or aGTP and the “test” agent; and detecting the formation of tubulinmonomers, dimers, or oligomers. The microtubule can be labeled with anyof a variety of labels, however in a preferred embodiment, it is labeledwith DAPI. The formation of tubulin monomers, dimers, or oligomers canbe detected by any of a wide variety of methods including, but notlimited to changes in DAPI fluorescence, fluorescent resonance energytransfer (FRET), centrifugation, and the like. The microtubules arepreferably microtubules that are either naturally stable (e.g., axonemalmicrotubules) or microtubules that have been stabilized (e.g., bycontact with an agent such as paclitaxel, a paclitaxel analogue, or anon-hydrolyzable nucleotide GTP analogue such asguanylyl-(α,β)-methylene diphosphate (GMPCPP)).

The assays can be run in solution or in solid phase (i.e. where one ormore assay components are attached to a solid surface. In oneembodiment, of solid-phase assays, the microtubule is attached to thesurface e.g., by direct binding or by binding with an agent such as aninactivated microtubule motor protein, an avidin-biotin linkage, ananti-tubulin antibody, a microtubule binding protein (MAP), or apolylysine. The microtubule severing protein or microtubuledepolymerizing protein is preferably a katanin, a p60 subunit of akatanin, an XKCM1, or an OP18 polypeptide. In a particularly preferredembodiment, the microtubule severing protein is a katanin or a p60subunit of a katanin as described herein.

It was also a discovery of this invention that the katanin p60 subunitexhibits both the ATPase and microtubule severing activity observed inkatanin. The p60 subunit thus provides a good target for screening forpotential therapeutic lead compounds. Thus, in another embodiment, thisinvention provides methods for screening and for identifying atherapeutic lead compound that modulates depolymerization or severing ofa microtubule system. The methods involve providing an assay mixturecomprising a katanin p60 subunit and a microtubule, contacting the assaymixture with a test compound to be screened for the ability to inhibitor enhance the microtubule-severing or ATPase activity of the p60subunit; and detecting specific binding of the test compound to said p60subunit or a change in the ATPase activity of the p60 subunit. Thedetecting can be by any of a wide variety of methods including, but notlimited to detecting ATPase activity using malachite green as adetection reagent. Binding activity can be easily detected in bindingassays in which the p60 subunit is labeled and said test agent isattached to a solid support or conversely, the test agent is labeled andthe p60 subunit is attached to a solid support. In a preferredembodiment, the ATPase assays are performed in the presence ofstabilized microtubules.

The assay methods of this invention are also amenable to high throughputscreening. Thus, in one embodiment, any of the methods described hereinis performed in an array where said array comprises a multiplicity ofreaction mixtures. each reaction mixture comprising a distinct anddistinguishable domain of said array, and wherein the assay steps areperformed in each reaction mixture. The array can take a number offormats, however, in one preferred format, the array comprises amicrotitre plate, preferably a microtitre plate comprising at least 48and more preferably at least 96 reaction mixtures. The test agent can beone of a plurality of agents and each reaction mixture can comprise oneagent of the plurality of agents.

In addition, this invention provides for polypeptides having microtubulesevering activity. The polypeptides comprise an isolated p60 subunit ofa katanin, where the p60 subunit is encoded by a nucleic acid thathybridizes under stringent conditions with a nucleic acid that encodesthe katanin p60 amino acid sequence (SEQ ID NO:1). In a particularlypreferred embodiment, the polypeptide is the polypeptide of SEQ ID NO:1or the polypeptide of SEQ ID NO: 1 having conservative substitutions.The polypeptide can comprise at least 8 contiguous amino acids from apolypeptide sequence encoded by a nucleic acid as set forth in SEQ IDNO:1, where the polypeptide, when presented as an antigen, elicits theproduction of an antibody that specifically binds to a polypeptidesequence encoded by a nucleic acid as set forth in SEQ ID NO:1; and thepolypeptide does not bind to antisera raised against a polypeptideencoded by a nucleic acid sequence as set forth in SEQ ID NO:1, that hasbeen fully immunosorbed with a polypeptide encoded by a nucleic acidsequence as set forth in SEQ ID NO:1. In a most preferred embodiment,the polypeptide is polypeptide of SEQ ID NO:1.

This invention also provides an isolated nucleic acid that encodes akatanin p60 subunit having microtubule severing activity. The nucleicacid preferably comprises a nucleic acid that specifically hybridizeswith a nucleic acid that encodes the polypeptide of SEQ ID NO:1 understringent conditions. The nucleic acid preferably encodes a polypeptideof SEQ ID NO:1 or conservative substitutions thereof. The katanin p60encoding nucleic acid can be operably linked to a promoter (e.g. abaculovirus promoter) and may be present in a vector.

In another embodiment, this invention provides methods of screening foran agent that alters microtubule polymerization, or depolymerization, orsevering. The methods involve providing labeled tubulin; contacting thelabeled tubulin with the test agent to produce contacted tubulin; andcomparing the fluorescence intensity or pattern of the contacted tubulinwith the fluorescence intensity or pattern of labeled tubulin that isnot contacted with the test agent where a difference in fluorescencepattern or intensity between the contacted and the not contacted tubulinindicates that the agent alters microtubule polymerization, ordepolymerization, or severing. In particularly preferred embodiments,the labeled tubulin is in the form of tubulin monomers, tubulin dimers,tubulin oligomers, or a microtubule. In some embodiments, themicrotubule is attached to a solid surface (e.g., by binding with anagent selected from the group consisting of an inactivated microtubulemotor protein, an avidin-biotin linkage, an anti-tubulin antibody, amicrotubule binding protein (MAP), a polyarginine, a polyhistidine, anda polylysine). Preferred labels include DAPI, ANS, Bis-ANS, rutheniumred, cresol violet, and DCVJ, with DAPI being most preferred. In someembodiments, the “contacting” step can further comprise contacting thetubulin with a microtubule depolymerizing protein or a microtubulesevering protein. Preferred microtubule severing or a microtubuledepolymerizing proteins include, but are not limited to katanin, a p60subunit of a katanin, an XKCM1, and a OP18 polypeptide. A preferred p60subunit of a katanin is a polypeptide of SEQ ID NO:1. The method canfurther involve listing the agents that alter microtubulepolymerization, depolymerization, or severing into a database oftherapeutic lead compounds that act on the cytoskeletal system. Thismethod can be performed in various array embodiments as describedherein.

This invention also provides kits for practice of any of the methodsdescribed herein. In one embodiment, the kits comprise one or morecontainers containing an isolated microtubule severing protein or amicrotubule depolymerizing protein. The kit can further comprise apolymerized microtubule labeled with DAPI. The microtubule can bestabilized by contact with paclitaxel or a paclitaxel derivative. Themicrotubule can also optionally be attached to a solid surface (e.g., bybinding with an inactivated motor protein). The microtubule severingprotein or microtubule depolymerizing protein is preferably selectedfrom the group consisting of a katanin, a p60 subunit of a katanin, anXKCM1, and a OP18 polypeptide. In a particularly preferred embodiment,the microtubule severing protein is a katanin or a p60 subunit of akatanin.

DEFINITIONS

The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form. Unless specifically limited, the term encompassesnucleic acids containing known analogues of natural nucleotides whichhave similar binding properties as the reference nucleic acid and aremetabolized in a manner similar to naturally occurring nucleotides.Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated.

Specifically, degenerate codon substitutions may be achieved bygenerating sequences in which the third position of one or more selected(or all) codons is substituted with mixed-base and/or deoxyinosineresidues (Batzer et al. (1991) Nucleic Acid Res. 19: 5081; Ohtsuka etal. (1985) J. Biol Chem. 260: 2605-2608; Cassol et al. (1992); andRossolini et al, (1994) Mol Cell Probes 8: 91-98). The term nucleic acidis used interchangeably with gene, cDNA, and mRNA encoded by a gene.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to designate a linear series of amino acidresidues connected one to the other by peptide bonds between thealpha-amino and carboxy groups of adjacent residues. The amino acidresidues are preferably in the natural “L” isomeric form. However,residues in the “D” isomeric form can be substituted for any L-aminoacid residue, as long as the desired functional property is retained bythe polypeptide. In addition, the amino acids, in addition to the 20“standard” amino acids, include modified and unusual amino acids, whichinclude, but are not limited to those listed in 37 CFR §1.822(b)(4).Furthermore, it should be noted that a dash at the beginning or end ofan amino acid residue sequence indicates either a peptide bond to afurther sequence of one or more amino acid residues or a covalent bondto a carboxyl or hydroxyl end group.

The term “conservative substitution” is used in reference to proteins orpeptides to reflect amino acid substitutions that do not substantiallyalter the activity (specificity or binding affinity) of the molecule.Typically conservative amino acid substitutions involve substitution oneamino acid for another amino acid with similar chemical properties (e.g.charge or hydrophobicity). The following six groups each contain aminoacids that are typical conservative substitutions for one another:

-   -   1) Alanine (A), Serine (S), Threonine (T);    -   2) Aspartic acid (D), Glutamic acid (E);    -   3) Asparagine (N), Glutamine (Q);    -   4) Arginine (R), Lysine (K);    -   5) Isoleucine (1), Leucine (L), Methionine (M), Valine (V); and    -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

The terms “isolated” and “biologically pure” refer to material which issubstantially or essentially free from components which normallyaccompany it as found in its native state. However, the term “isolated”is not intended to refer to the components present in an electrophoreticgel or other separation medium. An isolated component is free from suchseparation media and in a form ready for use in another application oralready in use in the new application/milieu.

The terms “identical,” percent “identity,” and percent “homology” in thecontext of two or more nucleic acids or polypeptide sequences, refer totwo or more sequences or subsequences that are the same or have aspecified percentage of amino acid residues or nucleotides that are thesame, when compared and aligned for maximum correspondence, as measuredusing one of the following sequence comparison algorithms or by visualinspection.

The phrase “substantially identical,” in the context of two nucleicacids or polypeptides, refers to two or more sequences or subsequencesthat have at least 60%, preferably 80%, most preferably 90-95% or evenat least 98% amino acid residue identity across a window of at least 30nucleotides, preferably across a window of at least 40 nucleotides, morepreferably across a window of at least 80 nucleotides, and mostpreferably across a window of at least 100 nucleotides, 150 nucleotides,200 nucleotides or greater, when compared and aligned for maximumcorrespondence, as measured using one of the following sequencecomparison algorithms or by visual inspection.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, forexample, by the local homology algorithm of Smith & Waterman, Adv. Appl.Math. 2:482 (1981), by the homology alignment algorithm of Needleman &Wunsch, J Mol. Biol. 48:443 (1970), by the search for similarity methodof Pearson & Lipman, Proc. Natl. Acad Sci. USA 85:2444 (1988), bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by visual inspection (Seegenerally, Ausubel et al., supra).

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments to show relationship and percent sequence identity.It also plots a tree or dendogram showing the clustering relationshipsused to create the alignment. PILEUP uses a simplification of theprogressive alignment method of Feng & Doolittle (1987) J. Mol. Evol. 35:3 51-360. The method used is similar to the method described byHiggins & Sharp (1989) CABIOS 5:151-153. The program can align up to 300sequences, each of a maximum length of 5,000 nucleotides or amino acids.The multiple alignment procedure begins with the pairwise alignment ofthe two most similar sequences, producing a cluster of two alignedsequences. This cluster is then aligned to the next most relatedsequence or cluster of aligned sequences. Two clusters of sequences arealigned by a simple extension of the pairwise alignment of twoindividual sequences. The final alignment is achieved by a series ofprogressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. For example, a reference sequence can be compared to othertest sequences to determine the percent sequence identity relationshipusing the following parameters: default gap weight (3.00), default gaplength weight (0.10), and weighted end gaps.

Another example of algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al. (1990) J. Mol Biol. 215:403-410.Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information. This algorithm involvesfirst identifying high scoring sequence pairs (HSPs) by identifyingshort words of length “W” in the query sequence, which either match orsatisfy some positive-valued threshold score “T” when aligned with aword of the same length in a database sequence. “T” is referred to asthe neighborhood word score threshold (Altschul et al, supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are then extended inboth directions along each sequence for as far as the cumulativealignment score can be increased. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity “X” from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters “W,” “T,” and “X” determine thesensitivity and speed of the alignment. The BLAST program uses asdefaults a wordlength (“W”) of 11, the BLOSUM62 scoring matrix (SeeHenikoff & Henikoff (1989) Proc. Natl. Acad Sci USA 89: 10915)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (See, e.g., Karlin & Altschul (1993) Proc. Natl. Acad Sci.USA 90: 5873-5787). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

A further indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid, as described below. Thus, apolypeptide is typically substantially identical to a secondpolypeptide, for example, where the two peptides differ only byconservative substitutions. Another indication that two nucleic acidsequences are substantially identical is that the two moleculeshybridize to each other under stringent conditions, as described below.

The phrases “hybridizing specifically to,” “specific hybridization,” and“selectively hybridize to,” refer to the binding, duplexing, orhybridizing of a nucleic acid molecule preferentially to a particularnucleotide sequence under stringent conditions when that sequence ispresent in a complex mixture (e.g., total cellular) DNA or RNA.

The term “stringent conditions” refers to conditions under which a probewill hybridize preferentially to its target subsequence, and to a lesserextent to, or not at all to, other sequences. Stringent hybridizationand stringent hybridization wash conditions in the context of nucleicacid hybridization experiments such as Southern and Northernhybridizations are sequence dependent, and are different under differentenvironmental parameters. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes part I chapter 2 Overview of Principles of Hybridization and theStrategy of Nucleic Acid Probe Assays, Elsevier, New York. Generally,highly stringent hybridization and wash conditions are selected to beabout 5° C. lower than the thermal melting point (T_(m)) for thespecific sequence at a defined ionic strength and pH. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Very stringentconditions are selected to be equal to the T_(m) for a particular probe.

An example of stringent hybridization conditions for hybridization ofcomplementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or Northern blot is 50% formamidewith 1 mg of heparin at 42° C., with the hybridization being carried outovernight. An example of highly stringent wash conditions is 0.15 M NaClat 72° C. for about 15 minutes. An example of stringent wash conditionsis a 0.2×SSC wash at 65° C. for 15 minutes (See, Sambrook et al. (1989)Molecular Cloning—A Laboratory Manual (2nd ed.) Vol. 1-3, Cold SpringHarbor Laboratory, Cold Spring Harbor Press, NY, for a description ofSSC buffer). Often, a high stringency wash is preceded by a lowstringency wash to remove background probe signal. An example mediumstringency wash for a duplex (e.g., of more than 100 nucleotides), is1×SSC at 45° C. for 15 minutes. An example low stringency wash for aduplex (e.g., of more than 100 nucleotides), is 4-6×SSC at 40° C. for 15minutes. In general, a signal to noise ratio of 2× (or higher) than thatobserved for an unrelated probe in the particular hybridization assayindicates detection of a specific hybridization. Nucleic acids which donot hybridize to each other under stringent conditions are stillsubstantially identical if the polypeptides which they encode aresubstantially identical. This occurs, for example, when a copy of anucleic acid is created using the maximum codon degeneracy permitted bythe genetic code.

The terms “katanin” or “katanin p60 subunit” refer to katanin and thekatanin p60 subunit as described herein, in the references cited and inthe sequence listings. The terms also include proteins havingsubstantial amino acid sequence identity with katanin or the katanin p60subunit sequences provided herein that exhibit ATPase and microtubulesevering activity.

The terms “taxol” and “taxol derivatives or analogues refer to the drugtaxol known generically as paclitaxel (NSC number: 125973). Paclitaxel(taxol) derivatives and analogues show similar microtubule-stabilizingactivity. Preferred derivatives include taxotere and others.

Depolymerized microtubule components are defined so as to include theproducts of microtubule depolymerization or severing, and includetubulin monomers, dimers and oligomers.

The term “test agent” refers to an agent that is to be screened in oneor more of the assays described herein. The agent can be virtually anychemical compound. It can exist as a single isolated compound or can bea member of a chemical (e.g., combinatorial) library. In a particularlypreferred embodiment, the test agent will be a small organic molecule.

The term small organic molecules refers to molecules of a sizecomparable to those organic molecules generally used in pharmaceuticals.The term excludes biological macromolecules (e.g., proteins, nucleicacids, etc.). Preferred small organic molecules range in size up toabout 5000 Da, more preferably up to 2000 Da, and most preferably up toabout 1000 Da.

The terms “label” or “detectable label” are used herein to refer to anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Such labelsinclude biotin for staining with labeled streptavidin conjugate,magnetic beads (e.g., Dynabeads™), fluorescent dyes (e.g., fluorescein,texas red, rhodamine, green fluorescent protein, and the like),radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase and others commonly used in anELISA), and colorimetric labels such as colloidal gold or colored glassor plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.Patents teaching the use of such labels include U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and4,366,241. Means of detecting such labels are well known to those ofskill in the art. Thus, for example, radiolabels may be detected usingphotographic film or scintillation counters, fluorescent markers may bedetected using a photodetector to detect emitted light. Enzymatic labelsare typically detected by providing the enzyme with a substrate anddetecting, the reaction product produced by the action of the enzyme onthe substrate, and colorimetric labels are detected by simplyvisualizing the colored label.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A (SEQ ID NO:1) and 1B (SEQ ID NOS: 4-9) show the sequenceanalysis of p60 katanin. FIG. 1A: Predicted protein sequence of the S.purpuratus katanin p60 subunit (SEQ ID NO:1; GENBANK AF052191).Sequences obtained by direct peptide microsequencing are underlined.Differences between the predicted peptide sequence and that obtained bydirect sequencing are indicated by doubled underlines (S95 was reportedas F, H99 was reported as P, and P138 was reported as T). The Walker A(P-loop) motif is shaded. FIG. 1B: Amino acid sequence alignment of thep60 AAA domain (SEQ ID NO:4) with AAA members mei-1 (SEQ ID NO:5; C.elegans, GenBank L25423), Suglp (SEQ ID NO:6; S. cerevisiae, GenBankX66400), ftsH (SEQ ID NO:7; E. coli, GenBank M83138), Paslp (SEQ IDNO:8; S. cerevisiae, GenBank M58676), and NSF (SEQ ID NO:9; C.longicaudatus, GenBank X15652). Identical residues are shaded black,residues conserved in >60% of the shown members are shaded gray. Lefthand numbering indicates the amino acid residue in the correspondingsequence. Alignment was performed using PILEUP (Genetics Computer Group)and the output was shaded using MACBOXSHADE.

FIG. 2A (SEQ ID NO:2) and 2B (SEQ ID NOS: 10-13) show the sequenceanalysis of p80 katanin. FIG. 2A: Predicted protein sequence of the S.purpuratus katanin p80 subunit (SEQ ID NO:2; GENBANK AF052433).Sequences obtained by direct peptide microsequencing are underlined.Differences between the predicted peptide sequence and that obtained bydirect peptide sequencing, or differences found between 2 different p80cDNA clones are indicated by double underlines. FIG. 2B: Amino acidsequence alignment of the WD40 repeat region of p80 (SEQ ID NO:10) witha putative human ortholog of p80 (SEQ ID NO:11; Homo sapiens p80,GenBank AF052432), TFIID (SEQ ID NO:12; Homo sapiens, GenBank U80191),and putative serine/threonine kinase PkwA (SEQ ID NO:13; Thermomonosporacurvata, GenBank P49695). Identical residues are shaded black, residuesfound in at least 2 sequences are shaded in grey. Left hand numberingindicates the amino acid residue in the corresponding sequence.Alignment was performed using PILEUP (Genetics Computer Group) and theoutput was shaded using MACBOXSHADE.

FIG. 3 illustrates the results of expression and purification ofrecombinant katanin subunits. Panel A shows Coomassie-stained SDS-PAGEanalysis of expressed katanin subunits. 6xHis-tagged (SEQ ID NO:14)katanin subunits were purified from lysates of baculovirus-infectedinsect cells by binding to Ni²⁺-NTA Superflow followed by elution withimidazole, as described in the Experimental Procedures. Cells wereinfected with either p60 virus alone, p80 virus alone, or coinfectedwith equal amounts of p60 and p80 viruses. Panel B showsimmunoprecipitation performed on extracts of insect cells coinfectedwith p60- and p80expressing baculoviruses using affinity-purified p60antibody crosslinked to protein A agarose. Proteins bound to the resinwere analyzed by SDS-PAGE followed by staining with Coomassie. Thisimmunoprecipitate shows that baculovirus-expressed p60 and p80 form acomplex with equal stoichiometry.

FIG. 4 shows the structure of katanin as visualized by rotary-shadowingelectron microscopy. Panel A shows 14-16 nm diameter rings observed inpreparations of recombinant p60. Panel B shows single particles ofrecombinant p80; occasional aggregates are seen (rightmost picture) butrings are never observed. Panels C and D show different rings observedin recombinant p60/p80 preparations. Panel C shows a “splayed” complex,consisting of a central p60-like ring surrounded by a halo particlesthat resemble p80. In Panel D, intact 20 nm diameter rings are seen withbright edges, suggesting they extend >10 nm above the mica surface. Allimages are shown at 300,000×. The dimensions indicated above include theplatinum shadowing, which typically adds 2 nm of material to the proteinsurface.

FIGS. 5A, 5B, and 5C illustrate the activities of recombinant kataninsubunits. FIG. 5A shows ATPase activities of 0.04 MM p60 katanin(squares) and co-expressed p60/p80 (circles) determined at variousmicrotubule concentrations as described in the Experimental Procedures.Both p60 katanin and p60/p80 show similar patterns of microtubulestimulation, with p60 katanin having approximately one half of themaximally stimulated ATPase activity of p60/p80. The insert in the upperright shows the stimulation of ATPase activity at low (0-2 μM)microtubule concentration. FIG. 5B shows microtubule severing activityof recombinant katanin subunits. Taxol-stabilized, rhodamine-labeledmicrotubules were adsorbed onto the surface of a microscope perfusionchamber, and then recombinant katanin subunits were introduced. The timeelapsed after perfusing p60/p80 (0.1 μM), p60 (0.1 μM), or p80 (0.5 μM)is shown. The recombinant co-expressed p60/p80 and p60, but not p80, cansever and disassemble microtubules. Scale bar, 10 μm. FIG. 5C showsquantitative measurement of microtubule disassembly using a DAPIfluorescence assay. MT indicates microtubules (2 μM) without addedprotein, and tubulin indicates microtubules that had been depolymerizedby treatment with 10 mM CaCl₂ on ice for 1.5 hr. p60 katanin and p60/p80were added at 0.2 μM concentration, and the fluorescence change as afunction of time after protein addition is shown. p80 did not cause achange in fluorescence that was different from that shown formicrotubules alone.

FIG. 6 shows that the WD40 repeats of p80 katanin are not required forinteraction with p60 katanin. Epitope-tagged derivatives of p80 and p60were synthesized in vitro in a combined transcription-translationreaction. p60 and interacting proteins were immunoprecipitated with ap60-specific antibody and the resulting immunoprecipitates were resolvedby SDS-PAGE and blotted to nitrocellulose. In vitro translated proteinswere detected by chemiluminescence as described in ExperimentalProcedures. Lane 1: molecular weight standards Mr: 100,000, 75,000,50,000, 35,000 and 25,000; lane 2: p60 co-translated with full-lengthp80; lane 3: p60 co-translated with the Δ560-690 derivative of p80; lane4: p60 co-translated with the Δ1-302 derivative of p80; lane 5: p60co-translated with the Δ303-690 derivative of p80. The structure of eachdeletion derivative of p80 is shown at right. The Δ560-690 and Δ303-690translation products were detected in the supernatants of theimmunoprecipitations (not shown).

FIG. 7 shows that human p80 katanin and a fusion protein of the humanp80 WD40 domain with GFP co-localize with γ-tubulin at centrosomes ofMSU1.1 human fibroblasts. Panels A and B: Co-localization ofimmunofluorescence staining by a human p80 katanin-specific antibody(Panel A) and a γ-tubulin specific antibody (Panel B). Panels C-F showco-localization of GFP fluorescence (Panels C and E) with staining by aγ-tubulin-specific antibody (Panels D and F). Co-localization to twocentrosomes seen in Panel C and Panel D while co-localization to asingle centrosome is seen in Panels E and F. The apparently higherbackground of cytoplasmic green fluorescence in Panel E relative toPanel C is a display artifact. The fluorescence intensity of thecentrosomes in Panel C is at least 5 fold greater than that of thecentrosome in Panel E. The p80 antibody was detected with an OregonGreen 488 second antibody and the γ-tubulin antibody was detected with aTexas Red-X second antibody. Fluorescence signals were separated withfluorescein and Texas Red filter sets (Chroma Technologies). Bar =14 μm.

DETAILED DESCRIPTION

I. Introduction.

This invention provides assays for the identification of agents thatmodulate the activity of microtubule depolymerizing or severingproteins. The assays generally involve contacting a polymerizedmicrotubule with a microtubule severing or depolymerizing protein (e.g.,XKCM1, OP18, katanin, etc.) in the presence of a test agent and achemical energy source (e.g., ATP or GTP). The effect of the agent onthe depolymerization or severing of the microtubules is then detectedtypically by detecting the formation of microtubule degradationcomponents (e.g., tubulin monomers, tubulin dimers, or tubulinoligomers). Test agents that alter the amount and or rate ofdepolymerization or severing of microtubules as compared to one or morecontrol assays are identified as modulators of microtubuledepolymerizing or severing activity.

It was a discovery of this invention that certain proteins that eitherdepolymerize or sever microtubules, provide good targets for modulatorsof normal mitotic spindle formation. Without being bound by a particulartheory, it is believed that microtubule depolymerizing or severingactivity is critical for normal mitotic spindle formation and/orfunction. Agents that modulate (e.g., upregulate, downregulate, orcompletely inhibit) depolymerization or severing activity are expectedto have a significant activity on progression of the cell cycle. Thus,for example, inhibitors of microtubule depolymerization or severing willact as potent antimitotic agents.

Anti-mitotic agents are useful in a wide variety of contexts. Aspowerful anti-mitotics or anti-meiotics, the inhibitors of microtubuledepolymerizing or severing activity identified by the screening (assay)methods described herein, will have a wide variety of uses, particularlyin the treatment (e.g., amelioration) of pathological conditionscharacterized by abnormal cell proliferation. Such conditions include,but are not limited to: fungal infections, abnormal stimulation ofendothelial cells (e.g., atherosclerosis), solid tumors and tumormetastasis, benign tumors (for example, hemangiomas, acoustic neuromas,neurofibromas, trachomas, and pyogenic granulomas), vascularmalfunctions (e.g., arterio-venous malformations), abnormal woundhealing, inflammatory and immune disorders, Bechet's disease, gout orgouty arthritis, abnormal angiogenesis accompanying: rheumatoidarthritis, psoriasis, diabetic retinopathy, and other ocular angiogenicdiseases such as retinopathy of prematurity (retrolental fibroplasic),macular degeneration, corneal overgrowth, corneal graft rejection,neuroscular glaucoma, Oster Webber syndrome, and the like.

The inhibitors of microtubule depolymerization or severing will alsohave a variety of in vitro uses as well. For example, they can be usedto freeze cells in a particular stage of the cell cycle for a variety ofpurposes (e.g., in the preparation of samples for histologicalexamination), in the isolation of nucleic acids from a particular stageof the cell cycle, and so forth.

The modulators identified by the assays of this invention are preferablycharacterized by specificity to the target microtubule depolymerizing orsevering proteins or the pathways characteristic of the activity ofthese proteins. They therefor provide novel lead compounds for thedevelopment of highly specific inhibitors for depolymerizing and/ormicrotubule severing protein families and subfamilies, thus allowing forprecise chemical intervention.

IL Assays for the Detection of Microtubule Depolymerization Modulators.

A) Depolymerization Assay

In one embodiment, this invention provides assays for thedetection/identification of agents that have activity in modulating thedepolymerization or severing of microtubules. The assays generallyinvolve contacting a polymerized microtubule with a microtubule severingprotein or a microtubule depolymerizing protein in the presence of achemical energy source (e.g., ATP or GTP) and said agent; and detectingand/or quantifying the formation of microtubule degradation products(e.g., tubulin monomers). Agents that inhibit the activity of themicrotubule depolymerizing or severing proteins will inhibit thebreakdown of the polymerized microtubules thereby delaying the formationof or reducing the quantity of tubulin monomers or oligomers. Thus adecrease in the rate of formation or amount of tubulin monomer or anincrease in the ratio of tubulin polymer (microtubule) to tubulinmonomer indicates an inhibitory modulating effect of the agent.Conversely, an increase in the rate of formation or amount of tubulinmonomer or an increase in the ratio of tubulin polymer (microtubule) totubulin monomer indicates a microtubule stabilizing modulating effect ofthe agent.

The increase or decrease is determined by reference to one or morecontrols. A control is essentially an identical assay that either lacksthe test agent or contains a “reference” agent that has a knownactivity. Assays lacking any test agent whatsoever act as negativecontrols, while assays utilizing an agent that has known modulatingactivity act as positive controls.

In a preferred embodiment, the assay is scored as positive (i.e., theagent has activity modulating a microtubule depolymerizing or severingprotein) when there is a significant difference between the negativecontrol and test assay and/or when there is no significant differencebetween the positive control and test assay. The significant differenceis preferably a statistically significant difference, more preferably atleast about a 10% difference, and most preferably at least about a 20%,30%, 50% or 100% difference.

The assays can be performed in solution or in solid phase (i.e., withone or more components of the assay attached to a solid surface) asdescribed below. One particularly preferred embodiment is describedherein in Example I. The various components of the assay are describedbelow.

B) Binding Assays.

In another embodiment, this invention provides binding assays toidentify agents that inhibit binding of depolymerizing or severingproteins to microtubules or for agents that specifically bind to themicrotubule depolymerizing or severing polypeptide or polypeptidesubunit.

In preferred binding assays, the ability of the test agent tospecifically bind to the depolymerizing or severing protein is assayed.In a particularly preferred embodiment, the ability of the test agent tospecifically bind to a katanin p60 domain is assayed.

There are a wide variety of formats for binding assays. In oneembodiment, the depolymerizing or severing protein or protein subunit isimmobilized on a surface and contacted with the test agent or converselytest agent(s) are immobilized on a surface and specific binding of theprotein or protein subunit is assayed. Binding is most easily detectedwhere the moiety in solution (test agent or depolymerizing or severingprotein) is labeled and after the contacting and washing off of unboundagents, identification of the labeled moiety associated with the supportsuggests binding.

Solution phase binding assays are also known to those of skill in theart. For example, in one embodiment, the binding assay is acosedimentation assay. In this (pelleting) assay, when the test agentbinds to the microtubule severing or depolymerizing protein or proteinsubunits, the bound agent and protein will cosediment when centrifuged.Unbound polypeptide and test agent will either sediment at a differentrate or remain fully in solution.

Methods of performing various binding assays can be found in copendingapplication U.S. Ser. No. 60/057,895 filed on Sep. 4, 1997. For ageneral description of different formats for protein binding assays,including competitive binding assays and direct binding assays (See,Stites and A. Terr (1991) Basic and Clinical Immunology, 7th Edition;Maggio (1980) Enzyme Immunoassay, CRC Press, Boca Raton, Fla.; andTijssen (1985) Practice and Theory of Enzyme Immunoassays, in LaboratoryTechniques in Biochemisty and Molecular Biology, Elsevier SciencePublishers, B.V. Amsterdam).

C) ATPase Assay.

It was a discovery of this invention that the katanin p60 subunit is anew member of the AAA family of ATPases and that expressed p60 hasmicrotubule-stimulated ATPase and microtubule-severing activities in theabsence of the p80 subunit. Thus, in another embodiment, this inventionprovides assays for agents that modulate the ATPase activity of akatanin p60 subunit.

ATPase assays are well known to those of skill in the art. In onepreferred embodiment, the assay can be performed according to themethods described by Kodama et al. (1986) J Biochem. 99: 1465-1472. Thisassay, described in detail in Example 1, is performed with the testagent present and the results are compared to negative and/or positivecontrol assays to determine the ability of the test agent to alter(modulate) p60 ATPase activity.

D) Solid Phase Assays.

In one embodiment, the assays of this invention can be performed insolid phase where one or more components of the assay is attached to asolid surface. In solid phase assays, one or more components of theassay is attached to a solid surface. Virtually any solid surface issuitable, as long as the surface material is compatible with the assayreagents and it is possible to attach the component to the surfacewithout unduly altering the reactivity of the assay components. It isrecognized that some components show reduced activity in solid phase,but this is generally acceptable so long as the activity is sufficientto detect and/or quantify depolymerization or severing activity of thesubject protein.

Solid supports include, essentially any solid surface, such as a glassbead, planar glass, controlled pore glass, plastic, porous plasticmetal, or resin to which the molecule may be adhered. One of skill willappreciate that the solid supports may be derivatized with functionalgroups (e.g., hydroxyls, amines, carboxyls, esters, and sulfhydryls) toprovide reactive sites for the attachment of linkers or the directattachment of the component(s).

Adhesion of the assay component (e.g., microtubule(s)) to the solidsupport can be direct (i.e., the microtubule directly contacts the solidsupport) or indirect (i.e., a particular compound or compounds are boundto the support, and the assay component binds to this compound orcompounds rather than to the solid support). The component can beimmobilized either covalently (e.g., utilizing single reactive thiolgroups of cysteine for anchoring protein components (Colliuod et al.(1993) Bioconjugate Chem. 4, 528-536)), or non-covalently butspecifically (e.g., via immobilized antibodies or other specific bindingproteins (Schuhmann et al. (1991), Adv. Mater. 3: 388-391; and Lu et al.(1995), Anal. Chem. 67: 83-87), the biotin/streptavidin system (Iwane etal. (1997) Biophys. Biochem. Res. Comm. 230: 76-80), or metal-chelatingLangmuir-Blodgett films (Ng et al. (1995) Langmuir 11: 4048-4055;Schmitt et al. (1996) Angew. Chem. Int. Ed. Engl. 35: 317-320; Frey etal. (1996) Proc. Nat. Acad. Sci. USA 93:4937-4941; and Kubalek et al.(1994) J Struct. Biol. 113:117-1231) and metal-chelating self-assembledmonolayers (Sigal et al. (1996) Analytical Chem., 68: 490-497) forbinding of polyhistidine fusion proteins.

In a preferred embodiment, the microtubule(s) are immobilized byattachment to an inactivated microtubule motor protein, by an avidinbiotin linkage (preferably with the biotin on the microtubule and theavidin on the surface), by an anti-tubulin antibody, by a microtubulebinding protein (MAP), by an amino silane, a polylysine, or throughinteraction with a polycationic surface.

By manipulating the solid support and the mode of attachment of theassay component to the support, it is possible to control theorientation of the assay component(s). For example, copendingapplication U.S. Ser. No. 60/057,929, filed on Sep. 4, 1997, describesthe use of an arginine tail to attach cytoskeletal proteins to a micafilm.

In one preferred embodiment, the microtubules are immobilized by coatingthe surface (e.g., a flow cell) with either n-ethylmaleimide(NEM)-treated Xenopus egg extract (6 mg/ml protein treated with 10 mMNEM for 10 minutes followed by addition of 100 mM dithiothreitol, atreatment that inactivates severing activity) or Escherichiacoli-expressed KAR3 protein (which binds microtubules in anucleotide-independent manner). After washing out unbound protein, thestabilized microtubules (e.g., 100 μg/ml in BR80, 20 μM taxol) areperfused onto the surface and allowed to bind. After washing out unboundmicrotubules samples to be tested can be contacted to the surface (e.g.,perfused into a flow cell).

E) High-throughput Screening of Candidate Agents that ModulateMicrotubule Depolymerizing or Severing Proteins.

Conventionally, new chemical entities with useful properties aregenerated by identifying a chemical compound (called a “lead compound”)with some desirable property or activity, creating variants of the leadcompound, and evaluating the property and activity of those variantcompounds. However, the current trend is to shorten the time scale forall aspects of drug discovery. Because of the ability to test largenumbers quickly and efficiently, high throughput screening (HTS) methodsare replacing conventional lead compound identification methods.

In one preferred embodiment, high throughput screening methods involveproviding a library containing a large number of potential therapeuticcompounds (candidate compounds). Such “combinatorial chemical libraries”are then screened in one or more assays, as described herein, toidentify those library members (particular chemical species orsubclasses) that display a desired characteristic activity. Thecompounds thus identified can serve as conventional “lead compounds” orcan themselves be used as potential or actual therapeutics.

i) Combinatorial Chemical Libraries

Recently, attention has focused on the use of combinatorial chemicallibraries to assist in the generation of new chemical compound leads. Acombinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biological synthesisby combining a number of chemical “building blocks” such as reagents.For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks called amino acids in every possible way for a given compoundlength (i.e., the number of amino acids in a polypeptide compound).Millions of chemical compounds can be synthesized through suchcombinatorial mixing of chemical building blocks. For example, onecommentator has observed at the systematic, combinatorial mixing of 100interchangeable chemical building blocks results in the theoreticalsynthesis of 100 million tetrameric compounds or 10 billion pentamericcompounds (Gallop et al. (1994) 37(9): 1233-1250).

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (See, e.g.,U.S. Pat. No. 5,010,175, Furka (1991) Int. J Pept. Prot. Res., 37:487-493; and Houghton et al. (1991) Nature, 354: 84-88). Peptidesynthesis is by no means the only approach envisioned and intended foruse with the present invention. Other chemistries for generatingchemical diversity libraries can also be used. Such chemistries include,but are not limited to: peptoids (PCT Publication No WO 91/19735, 26Dec. 1991), encoded peptides (PCT Publication WO 93/20242, 14 Oct.1993), random bio-oligomers (PCT Publication WO 92/00091, 9 Jan. 1992),benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such ashydantoins, benzodiazepines and dipeptides (Hobbs et al., (1993) Proc.Nat. Acad Sci. USA 90: 6909-6913), vinylogous polypeptides (Hagihara etal. (1992) J Amer. Chem. Soc. 114: 6568), nonpeptidal peptidomimeticswith a Beta- D- Glucose scaffolding (Hirschmann et al., (1992) J. Amer.Chem. Soc. 114: 9217-9218), analogous organic syntheses of smallcompound libraries (Chen et al. (1994) J Amer. Chem. Soc. 116: 2661),oligocarbamates (Cho, et al., (1993) Science 261: 1303), and/or peptidylphosphonates (Campbell et al., (1994) J. Org. Chem. 59: 658); See,generally, Gordon et al., (1994) J. Med Chem. 37: 1385), nucleic acidlibraries (See, e.g., Strategene, Corp.), peptide nucleic acid libraries(See, e.g., U.S. Pat. No. 5,539,083) antibody libraries (See, e.g.,Vaughn et al. (1996) Nature Biotechnology, 14(3): 309-314), andPCT/US96/10287), carbohydrate libraries (See, e.g., Liang et al. (1996)Science, 274: 1520-1522, and U.S. Pat. No. 5,593,853), and small organicmolecule libraries (See, e.g., benzodiazepines, Baum (1993) C&EN, Jan18, page 33; isoprenoids U.S. Pat. No. 5,569,588; thiazolidinones andmetathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos.5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337;benzodiazepines, U.S. Pat. No. 5,288,514; and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (See, e.g., 357 NIPS, 390 NTS, Advanced Chem Tech, LouisvilleKy., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif, 9050 Plus, Millipore, Bedford, Mass.).

A number of well known robotic systems have also been developed forsolution phase chemistries. These systems include automated workstationslike the automated synthesis apparatus developed by Takeda ChemicalIndustries, LTD. Osaka, Japan) and many robotic systems utilizingrobotic arms (Zymate II, Zymark Corporation, Hopkinton, Mass.; Orca,Hewlett-Packard, Palo Alto, Calif.) which mimic the manual syntheticoperations performed by a chemist. Any of the above devices are suitablefor use with the present invention. The nature and implementation ofmodifications to these devices (if any) so that they can operate asdiscussed herein will be apparent to persons skilled in the relevantart. In addition, numerous combinatorial libraries are themselvescommercially available (See, e.g., ComGenex, Princeton, N.J., Asinex,Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3DPharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md., etc.).

ii) High Throughput Assays of Chemical Libraries

Any of the assays for compounds modulating the activity of microtubuledepolymerizing or severing proteins (or other agents) described hereinare amenable to high throughput screening. As described above, in apreferred embodiment, the assays screen for agents that enhance orinhibit the activity of katanin, XKCM1, or OP18. Preferred assays detectthe rate or amount of depolymerization of microtubules into tubulinmonomers, tubulin dimers, or tubulin oligomers.

High throughput implementation of the assays described herein can beimplemented with, at most, routine modification of the assays format(e.g.,. for compatibility with robotic manipulators, large platereaders, and the like). Various high throughput screening systems (e.g.,for protein binding, nucleic acid binding, etc.) are described in U.S.Pat. Nos. 5,559,410, 5,585,639, 5,576,220, and 5,541,061.

In addition, high throughput screening systems are commerciallyavailable (See, e.g., Zymark Corp., Hopkinton, Mass.; Air TechnicalIndustries, Mentor, Ohio; Beckman Instruments, Inc. Fullerton, Calif.;Precision Systems, Inc., Natick, Mass., etc.). These systems typicallyautomate entire procedures including all sample and reagent pipetting,liquid dispensing, timed incubations, and final readings of themicroplate in detector(s) appropriate for the assay. These configurablesystems provide high throughput and rapid start up as well as a highdegree of flexibility and customization. The manufacturers of suchsystems provide detailed protocols the various high throughput. Thus,for example, Zymark Corp. provides technical bulletins describingscreening systems for detecting the modulation of gene transcription,ligand binding, and the like.

III) Assay Components.

A) Polymerized Microtubules

As indicated above, the assays of this invention utilize polymerizedmicrotubules, which, in the presence of a depolymerizing protein orsevering protein are depolymerized or cleaved to produce tubulinmonomers or oligomers. When the depolymerizing or severing proteins areinhibited, the formation of tubulin monomers or oligomers is inhibited.

Virtually any microtubules can be used for the assays of this invention.Means of obtaining such microtubules are well known to those of skill inthe art. Tubulin is available commercially or can be isolated from awide variety of sources (e.g., plants, animal tissues, oocytes, etc.)For example, tubulin can be isolated from Arabidopsis cells instationary phase (day 10 to II) cultured cells (200 to 500 gm freshweight) by DEAE-Sephadex A50 chromatography as described by Morejohn etal. (1985). Cell Biol. Int. Rep. 9(9): 849-857 with modificationsdescribed in Bokros et al. (1993), Biochemistry 32(13): 3437-3447.Briefly, Arabidopsis cells are homogenized in an isolation buffer (IB)consisting of 50 mM PIPES-KOH, pH 6.9, 1 mM EGTA, 0.5 mM MgS0₄, 1 mM DTTand 0.1 mM GTP, supplemented with 50 mg/mL Na-p-tosyl-L-arginine methylester (TAME), and 5 mg/mL each of pepstatin A, leupeptin hemisulfate,and aprotinin. The cell homogenate is subjected to DEAE-Sephadex A50chromatography for tubulin isolation. Ammonium sulfate precipitates ofDEAE-isolated tubulin can be aliquoted and stored at -80° C. until use.

Microtubules can be purified to homogeneity by a single taxol-inducedmicrotubule polymerization step in IB supplemented with 1 mM GTP asdescribed previously (Bokros et al. (1993), Biochemistry 32(13):3437-47). Briefly, samples of DEAE-isolated tubulin are thawed andresuspended in IB supplemented with 1 mM DTT and 1 mM GTP, and clarifiedby centrifugation for 1 hr at 100,000× g (2° C.) in a Beckman TL-100ultracentrifuge (TLA-100 rotor). Clarified tubulin is polymerized with atwofold molar excess of taxol in a microtubule assembly buffer composedof IB, 1 mM DTT, 1 mM GTP and 1% DMSO. Assembly of microtubules wasperformed by gradual temperature ramping from 2° C. to 25° C. over a2-hour period. Polymer is collected by centrifugation for 45 min at30,000×g at 25° C. through a cushion of 20% (w/v) sucrose in assemblybuffer.

In a preferred embodiment, the tubulin/microtubules are isolated from ananimal tissue (e.g., brain tissue) according to the methods of Hyman etal. (1991) Meth Enzy., 196: 478-485. The brain tubulin can be modifiedwith tetramethylrhodamine or fluorescein N-hydroxysuccinimide ester(Molecular Probes, Inc., Eugene, as described by Hyman et al. (1991)supra.

Most microtubules are in a state of flux, undergoing assembly anddisassembly. In a preferred embodiment of the assays of this inventionmicrotubules are utilized that are stabilized as essentially intactmicrotubules. This can be accomplished by using microtubules that arenaturally stable (e.g., axonemal microtubules) or by treating themicrotubules so that they are stabilized. Methods of stabilizingmicrotubules are well known to those of skill in the art and include,but are not limited to the use of stabilizing agents such as paclitaxeland paclitaxel derivatives (e.g., taxotere), non-hydrolyzable nucleotide(e.g., GTP) analogues (e.g., guanylyl-(α,β)-methylene diphosphate(GMPCPP)), and the like.

In a preferred embodiment, taxol-stabilized microtubules, are preparedby polymerizing tubulin (2-10 mg/ml) at 37° C. for 45 minutes in BRB80(80 mM PIPES [pH 6.8], 1 mM MgCl₂, 1 mM EGTA) containing 1 mM GTP and10% dimethyl sulfoxide (DMSO). Taxol is then added to a concentration of20 μM.

GMPCPP-stabilized microtubules are prepared by incubating tubulin in theabove, buffer, substituting 0.5 mM GMPCPP for GTP. The GMPCPPmicrotubules can be stored at room temperature without taxol and arepreferably used within 1 day after preparation.

B) Assay Reaction Mixture.

The assays of this invention are performed in a reaction mixture thatprovides the components necessary for microtubule depolymerizing ormicrotubule severing activity of the subject protein (e.g., katanin,XKCM1, OP18 etc.) and that are compatible with the enzymatic activity ofthe subject proteins. Typically the reaction mixture comprises anappropriate buffer (e.g., HEPES, pH 6.5-8.0) and an energy supplyingmolecule such as guanosine triphosphate (GTP) for microtubuledepolymerizing proteins or adenosine triphosphate (ATP) for severingmolecules such as katanin. One preferred assay,mixture is described inExample 1.

C) Microtubule Depolymerizing and Microtubule Severing Agents.

As indicated above, the assays of this invention essentially detect theactivity of a test agent on a microtubule depolymerizing or microtubulesevering polypeptide. Microtubule depolymerizing polypeptides such asOP18 (Belmont et al. (1990) Cell, 62: 579-589) and XKCM1 (Walczak et al.(1996) Cell, 83: 37-47) increase the frequency of catastrophes(transitions of a microtubule from a growing to a shrinking state) andthus promote disassembly of microtubules from their ends.

In contrast to microtubule depolymerizing proteins, other proteins, suchas katanin, promote the disassembly of microtubules by generatinginternal breaks within a microtubule and are referred to as microtubulesevering proteins (See, e.g., Vale (1991) Cell 64: 827-839; Shiina etal. (1994) Science 266: 282-285; Shiina et al. (1992) EMBO J. 11:4723-4731; and McNally and Vale (1993) Cell, 75: 419-429).

Preferred microtubule depolymerizing proteins for the methods of thisinvention include, but are not limited to XKCM1, and OP18, whilepreferred microtubule severing proteins include katanin.

i) Katanin.

Katanin, a heterodimer of 60 kDa and 80 kDa subunits purified from seaurchin eggs, is unique among the known microtubule and actin severingproteins in that it disrupts contacts within the polymer lattice byusing energy derived from ATP hydrolysis (McNally and Vale (1993) Cell,75: 419-429). Katanin acts substoichiometrically, as one molecule ofkatanin can release several tubulin dimers from a microtubule. Katanindoes not appear to proteolyze or modify tubulin, since the tubulinreleased from the disassembly reaction is capable of repolymerizing(McNally and Vale (1993) Cell, 75: 419-429). The mechanism ofmicrotubule severing by katanin, however, is not understood.

Katanin-catalyzed microtubule severing and disassembly could potentiallybe involved in several changes in the microtubule cytoskeleton observedin vivo. Recent studies have-shown that katanin is concentrated at thecentrosome in a microtubule-dependent manner in sea urchin embryos(McNally et al. (1996) J Cell Sci. 109: 561-567). One phenomenon thatcould require disassembly of microtubules at the centrosome is thepoleward flux of tubulin in the mitotic spindle (Mitchison (1989) J CellBiol. 109: 637-652). The disassembly of microtubule minus ends at thespindle pole during poleward flux could be driven by katanin, or katanincould simply allow depolymerization by uncapping microtubule minus endsthat are docked onto γ-tubulin ring complexes (Zheng et al. (1995)Nature 378: 578-583; Moritz et al. (1995) Nature 378: 638-640). Anotherpossible role for katanin at the centrosome is in promoting the releaseof microtubules from their centrosomal attachment points. Microtubulesare nucleated from γ-tubulin ring complexes at the centrosome (Joshi etal., (1992) and Moritz et al. (1995) Nature 378: 63 8-640), but releaseof microtubule minus ends has been observed indirectly in Dictyostelium(Kitanishi-Yumura et al. (1987) Cell Motil. Cytoskeleton 8: 106-117) anddirectly in PtK1 cells (Keating (1997) Proc. Natl. Acad. Sci. USA, 94:5078-5083) and Xenopus egg extracts (Belmont et al. (1990) Cell 62:579-589). Finally, katanin could accelerate the rapid disassembly of theinterphase microtubule network at the G2/M transition (Zhai et al.(1996) J. Cell Biol. 135: 201-214) by severing cytoplasmic microtubules,which would increase the number of free microtubule ends from whichdepolymerization could occur. Regardless of the particular mode ofactivity, modulation of katanin activity will have profound effects onthe cell cycle.

The amino acid and nucleic acid sequences of the p60 and p80 subunits ofkatanin are provided in FIGS. 1A and 2A (See also SEQ ID NO:1 and SEQ IDNO:2). It was a discovery of this invention that the microtubulesevering activity resides entirely in the p60 subunit. Thus the assaysof this invention can be practiced either with the heterodimeric kataninor with a p60 subunit alone.

The p60 and/or p80 subunits of katanin can be purified (e.g., from seaurchin eggs, e.g., eggs from Strongylocentrotus purpuratus) as describedby McNally and Vale (1993) Cell, 75: 419-429. Alternatively, either orboth subunits can be recombinantly expressed and purified as describedbelow and in Example 1.

ii) XKCM1

XKCM1 (for Xenopus kinesin central motor 1) is a motor protein essentialfor mitotic spindle assembly in vitro. XKCM1 localizes to centromeresand appears to regulate the polymerization dynamics of microtubules. Theisolation of an XKCM1 clone is described by Walczak et al. (1996) Cell,84: 37-47, and a nucleic acid sequence of an XKCMl cDNA is providedtherein and in SEQ ID NO:3. Using this sequence information, XKCM1 canbe expressed as described below and by Walczak et al. (1996) supra.

iii) OP18

Another microtubule depolymerizing motor protein suitable for use in themethods of this invention is OP18, also called stathmin orstathmin/op18. OP18 is described in detail by Gradin et al. (1998) JCell Biol., 140(1): 131-141, by Andersen et al. (1997) Nature,389(6651): 640-643, by Larsson et al. (1997) Mol. Cell. Biol.,17(9):5530-5539, and by Belmont et al. (1996) Cell, 84(4): 623-631.

iv) Other Microtubule Severing or Depolymerizing Proteins

Other microtubule depolymerizing or severing proteins include, but arenot limited to elongation factor-1α(Shiina et al. (1994) Science 266:282-285) and a novel homo-oligomeric protein described by Shiina et al.(1992) EMBO J. 11: 4723-4731.

Other microtubule depolymerizing or severing proteins can be identifiedwith only routine experimentation. The assays used to identifymicrotubule depolymerizing or severing proteins are identical to theassays described herein, the only difference being that no test agent isrequired. A detailed example of the assay of a microtubule severingprotein (katanin) is provided in McNally and Vale (1993) Cell, 75:419-429. The same approach can readily be used to identify othersevering or depolymerizing proteins.

IV) Detection Methods.

Any detection method that allows detection and/or quantification of theamount or rate of appearance of tubulin monomers or oligomers and/or therate of disappearance of assembled (polymerized) microtubules can beused in the assays of this invention. Preferred detection methodsinclude, but are not limited to video microscopy; DAPI fluorescencechanges, fluorescence resonance energy transfer and centrifugation.

A) Video Microscopy.

In one embodiment, microtubule depolymerization or severing is detectedby microscopy (visually or using a video or photographic recordingdevice). Assays involving microscopic visualization of microtubulespreferably utilize labeled (e.g., fluorescently labeled) microtubules.The microtubules are preferably immobilized on a solid support (e.g., aglass slide), and exposed to a solution containing the microtubuledepolymerizing or severing protein and an a nucleoside triphosphate. Theintact and depolymerized or severed microtubules can be directlyvisualized using a microscope. Microtubule depolymerization in thecontrol and the assay containing the test agent can be visualized sideby side or sequentially.

The microscope can optionally be equipped with a still camera or a videocamera and may be equipped with image acquisition and analysis softwareto quantify the relative abundance of intact and fragmentedmicrotubules.

This method can be used with essentially any label that can bevisualized in a microscope. Such labels include, but are not limited tofluorescent labels (e.g., fluorescein, rhodamine, etc.), colorimetriclabels, and radioactive labels (with appropriate scintillation screen),and the like. In some embodiments of the assays of this invention, themicrotubules can be visualized without any label (e.g., via differentialinterference contrast microscopy).

An illustration of the use of video microscopy to visualize microtubulesevering by katanin is provided by McNally and Vale (1993) Cell, 75:419-429. In this case, the microtubules are labeled with rhodamine andthe images of the severed microtubules are captured digitally.

B) DAPI Fluorescence changes.

In another embodiment, the state of microtubule polymerization can bedetermined by changes in fluorescence of DAPI stained microtubules. Ithas been shown that DAPI fluorescence intensity is higher when this dyeis bound to polymerized versus free tubulin (Heusele et al. (1987) Eur.J Biochem. 165: 613-620). When katanin and ATP were incubated withDAPI-labeled microtubules, a linear decrease in fluorescence intensityis observed as a function of time, reflecting the conversion ofmicrotubules to tubulin.

Assay can thus be performed as described above with DAPI labeledstabilized microtubules. The rate or amount decrease in DAPIfluorescence is detected as described by Heusele et al. supra. Thechange in fluorescence with a test agent is compared to that observed ina negative and/or positive control reaction.

It was a surprising discovery of this invention that tubulin, tubulindimers, tubulin oligomers or microtubules can be labeled with variouslabels such as DAPI and that the label does not interfere with theinteraction of various test agents or cytoskeletal associated proteinswith the labeled tubulin to a degree that would prevent assaying theimpact of a test agent on microtubule polymerization, and/ordepolymerization, and/or severing. Labels that can be used include, butare not limited to anilinonapthalene sulfonate (ANS) (e.g., MolecularProbes Catalogue Nos: A-47, A-50, T-53, etc.), bis-ANS (Molecular ProbesCatalogue No: B-153), N-phenyl-1-naphthylene (NPN) (Molecular ProbesCatalogue No: P65), DCVJ (Molecular Probes Catalogue No: D-3923),ruthenium red, and cresol violet.

C) Fluorescence resonance energy transfer

The degree of microtubule polymerization/depolymnerization can also bedetermined by fluorescent resonance energy transfer (FRET). Fluorescenceresonance energy transfer, a phenomenon that occurs when twofluorophores with overlapping absorption and emission spectra arelocated close together (e.g., <7 nm apart) (Stryer (1978) Ann. Rev.Biochem., 47: 819-846). FRET is a powerful technique for measuringprotein-protein associations and has been used previously to measure thepolymerization of monomeric actin into a polymer (Taylor et al. (1981)J. Cell Biol., 89: 362-367) and actin filament disassembly by severing(Yamamoto et al. (1982) J. Cell Biol., 95: 711-719).

In a preferred embodiment, equimolar proportions of differently labeled(e.g., fluorescein labeled and rhodamine-labeled) tubulin are combined.The fluorescence is quenched upon tubulin polymerization indicating thatthe tubulin-bound fluorochromes in a microtubule come in close enoughproximity for energy transfer to occur. When the microtubule isdepolymerized or severed, a rapid unquenching of (e.g., fluorescein)fluorescence is observed.

The rates and/or amount of fluorescence generated by a reaction with atest agent and a control can be compared. A decrease in rate or amountof fluorescence in the presence of a test agent indicates inhibitoryactivity on the microtubule depolymerizing or severing protein(s).

In particularly preferred embodiment, the microtubules are polymerizedfrom a mixture of equal concentrations of fluorescein and rhodaminetubulin and diluted to 600 μg/ml tubulin in BRB80 containing 20 μM taxoland the oxygen-depleting system consisting of glucose oxidase (30μg/ml), catalase (100 mg/ml), glucose (10 mM), and dithiothreitol (10mM) (Kishino and Yanigida (1988) Nature 334: 74-76). Aliquots 150 μl ofthese microtubules can be mixed (e.g., with samples of purified severingprotein), and the fluorescence from the fluorescein (excitation 492 nm;emission 518 nm) is recorded. The reaction is preferably run with apositive and negative control. A detailed FRET assay for tubulinpolymerization is found in McNally et al. (1993) Cell, 75: 419-429.

D) Centrifugation.

Microtubule disassembly in solution can be documented in a quantitativemanner by examining the relative amounts of sedimentable andnonsedimentable tubulin after incubation with the severing ordepolymerizing protein (e.g., katanin) and ATP or GTP. In the absence ofa modulating agent, when taxol-stabilized microtubules are incubatedtithe the katanin p80 and p60 subunits and ATP, nonsedimentable tubulinis released from microtubule polymer in an approximately linear manner.The rate of release varies with microtubule depolymerizing or severingprotein concentration and will be dependent on the activity of amodulating “test” agent if present.

In a preferred embodiment, fluorescent microtubules (e.g., 100-300 μg/mltubulin) are incubated with the microtubule depolymerizing or severingprotein in buffer (e.g., 20 mM HEPES (pH 7.5), 2 mM MgCl₂, 25 mMpotassium glutamate, 0.02% Triton X-100, 250 μg/ml SBTI, and 20 μM taxolor taxol derivative) at various times. Aliquots (e.g., of 100 μl) arebrought to 10 mM ADP or GDP (to stop the severing or depolymerizingreaction) and sedimented (e.g., at 228,000×g for 10 minutes).Supernatants are removed, and pellets are resuspended in 100 μl ofbuffer. The pellets and supernatants can be brought up to 300 μl (e.g.,with BRB80), and the relative fluorescence signals in the supernatantand the pellet are quantitated using a Perkin Elmer L25B luminescencespectrometer.

E) Liquid Crystal Assay Systems.

In still another embodiment, binding of the microtubule depolymerizingor severing proteins to microtubules can be detected by the use ofliquid crystals. Alternatively, it is expected that liquid crystals canbe used to monitor the state of tubulin polymerization.

Liquid crystals can be used to amplify and transduce receptor-mediatedbinding of proteins at surfaces into optical outputs. Spontaneouslyorganized surfaces can be designed so that protein molecules, uponbinding to ligands (e.g., microtubules) hosted on these surfaces,trigger changes in the orientations of 1- to 20-micrometer-thick filmsof supported liquid crystals, thus corresponding to a reorientation of˜10⁵ to 10⁶ mesogens per protein. Binding-induced changes in theintensity of light transmitted through the liquid crystal are easilyseen with the naked eye and can be further amplified by using surfacesdesigned so that protein-ligand recognition causes twisted nematicliquid crystals to untwist (See, e.g., Gupta et al. (1998) Science, 279:2077-2080). This approach to the detection of ligand-receptor bindingdoes not require labeling of the analyte, does not require the use ofelectroanalytical apparatus, provides a spatial resolution ofmicrometers, and is sufficiently simple that it is useful in biochemicalassays and imaging of spatially resolved chemical libraries.

D) Synthesis or Expression of Microtubule Depolymerizing or SeveringProteins.

i) De Novo Chemical Synthesis.

Using the information provided, herein, the microtubule depolymerizingor severing proteins, protein subunits, or subsequences thereof may besynthesized using standard chemical peptide synthesis techniques. Wherethe desired subsequences are relatively short, the molecule may besynthesized as a single contiguous polypeptide. Where larger moleculesare desired, subsequences can be synthesized separately (in one or moreunits) and then fused by condensation of the amino terminus of onemolecule with the carboxyl terminus of the other molecule therebyforming a peptide bond.

Solid phase synthesis in which the C-terminal amino acid of the sequenceis attached to an insoluble support followed by sequential addition ofthe remaining amino acids in the sequence is the preferred method forthe chemical synthesis of the polypeptides of this invention. Techniquesfor solid phase synthesis are described by Barany and Merrifield,Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis,Synthesis, Biology. VOL 2: Special Methods in Peptide Synthesis, PartA.; Merrifield, et al. (1963) J. Am. Chem. Soc., 85: 2149-2156; andStewart et al (1984) Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem.Co., Rockford, Ill.

ii) Recombinant Expression.

In a preferred embodiment, the microtubule depolymerizing or severingproteins, protein subunits, or subsequences, are synthesized usingrecombinant DNA methodology. Generally this involves creating a DNAsequence that encodes the protein, placing the DNA in an expressioncassette under the control of a particular promoter, expressing, theprotein in a host, isolating the expressed protein and, if required,renaturing the protein. DNA encoding the microtubule depolymerizing orsevering proteins, protein subunits, or subsequences of this inventioncan be prepared by any suitable method as described above, including,for example, cloning and restriction of appropriate sequences or directchemical synthesis by methods such as the phosphotriester method ofNarang et al. (1979) Meth. Enzymol. 68: 90-99; the phosphodiester methodof Brown et al. (1979) Meth. Enzymol. 68: 109-151; thediethylphosphoramidite method of Beaucage et al. (1981) Tetra. Lett.,22: 1859-1862; and the solid support method of U.S. Pat. No. 4,458,066.

Chemical synthesis produces a single stranded oligonucleotide. This maybe converted into double stranded DNA by hybridization with acomplementary sequence, or by polymerization with a DNA polymerase usingthe single strand as a template. One of skill would recognize that whilechemical synthesis of DNA is limited to sequences of about 100 bases,longer sequences may be obtained by the ligation of shorter sequences.

Alternatively, subsequences may be cloned and the appropriatesubsequences cleaved using appropriate restriction enzymes, Thefragments may then be ligated to produce the desired DNA sequence.

In one embodiment, the microtubule depolymerizing or severing proteins,of this invention can be cloned using DNA amplification methods such aspolymerase chain reaction (PCR). Thus, for example, the nucleic acidsequence or subsequence is PCR amplified, using a sense primercontaining one restriction site (e.g., NdeI) and an antisense primercontaining another restriction site (e.g., HindIll). This will produce anucleic acid encoding the desired the microtubule depolymerizing orsevering protein having terminal restriction sites. This nucleic acidcan then be easily ligated into a vector containing a nucleic acidencoding the second molecule and having the appropriate correspondingrestriction sites.

Suitable PCR primers can be determined by one of skill in the art usingthe sequence information provided herein. Appropriate restriction sitescan also be added to the nucleic acid encoding the microtubuledepolymerizing or severing proteins by site-directed mutagenesis. Theplasmid containing the microtubule depolymerizing or severing proteinencoding nucleic acid is cleaved with the appropriate restrictionendonuclease and then ligated into the vector encoding the secondmolecule according to standard methods.

The nucleic acid sequences encoding the microtubule depolymerizing orsevering proteins may be expressed in a variety of host cells, includingE. coli, other bacterial hosts, yeast, and various higher eukaryoticcells such as the COS, CHO and HeLa cells lines and myeloma cell lines.As the microtubule depolymerizing or severing proteins are typicallyfound in eukaryotes, a eukaryote host is preferred. The recombinantprotein gene will be operably linked to appropriate expression controlsequences for each host. For E. coli, this includes a promoter such asthe T7, trp, or lambda promoters, a ribosome binding site and preferablya transcription termination signal. For eukaryotic cells, the controlsequences will include a promoter and preferably an enhancer derivedfrom immunoglobulin genes, SV40, cytomegalovirus, etc., and apolyadenylation sequence, and may include splice donor and acceptorsequences.

The plasmids of the invention can be transferred into the chosen hostcell by well-known methods such as calcium chloride transformation forE. coli and calcium phosphate treatment or electroporation for mammaliancells. Cells transformed by the plasmids can be selected by resistanceto antibiotics conferred by genes contained on the plasmids, such as theamp, gpt, neo and hyg genes.

Once expressed, the recombinant the microtubule depolymerizing orsevering proteins can be purified according to standard procedures ofthe art, including ammonium sulfate precipitation, affinity columns,column chromatography, gel electrophoresis and the like (See generally,R. Scopes, (1982) Protein Purification, Springer-Verlag, N.Y.; Deutscher(1990) Methods in Enzymology Vol. 182. Guide to Protein Purification.,Academic Press, Inc. N.Y.). Substantially pure compositions of at leastabout 90 to 95% homogeneity are preferred, and 98 to 99% or morehomogeneity are most preferred. Once purified, partially or tohomogeneity as desired, the polypeptides may then be used (e.g., asimmunogens for antibody production).

One of skill in the art would recognize that after chemical synthesis,biological expression, or purification, the microtubule depolymerizingor severing protein(s) may possess a conformation substantiallydifferent than the native conformations of the constituent polypeptides.In this case, it may be necessary to denature and reduce the polypeptideand then to cause the polypeptide to re-fold into the preferredconformation. Methods of reducing and denaturing proteins and inducingre-folding are well known to those of skill in the art (See, Debinski etal. (1993) J. Biol. Chem., 268: 14065-14070; Kreitman and Pastan (1993)Bioconjug. Chem., 4: 581-585; and Buchner, et al., (1992) Anal.Biochem., 205: 263-270). Debinski et al., for example, describes thedenaturation and reduction of inclusion body proteins in guanidine-DTE.The protein is then refolded in a redox buffer containing oxidizedglutathione and L-arginine.

One of skill would recognize that modifications can be made to themicrotubule depolymerizing or severing proteins without diminishingtheir biological activity. Some modifications may be made to facilitatethe cloning, expression, or incorporation of the targeting molecule intoa fusion protein. Such modifications are well known to those of skill inthe art and include, for example, a methionine added at the aminoterminus to provide an initiation site, or additional amino acids (e.g.,polyHis) placed on either terminus to create conveniently locatedrestriction sites or termination codons or purification sequences.

In a particularly preferred embodiment, the katanin protein(s) areexpressed as described in Example 1, while XKCM1 is expressed andpurified as described by Walczak et al. (1996) supra.

V. Data Management.

In one embodiment, the assays of this invention are facilitated by theuse of databases to record assay results. Particular with the use oflarge-scale screening systems, (e.g., screening of combinatoriallibraries) data management can become a significant issue. For example,all natural hexapeptides have been synthesized in a single combinatorialexperiment producing about 64 million different molecules. Maintenanceand management of even a small fraction of the information obtained byscreening such a library is aided by methods automated informationretrieval (e.g., a computer database).

Such a database is useful for a variety of functions, including, but notlimited to library registration, library or result display, libraryand/or result specification, documentation, and data retrieval andexploratory data analysis. The registration function of a databaseprovides recordation/registration of combinatorial mixtures and assayresults to protect proprietary information in a manner analogous to theregistration/protection of tangible proprietary substances. Library andassay result display functions provide an effective means to reviewand/or categorize relevant assay data. Where the assays utilize complexcombinatorial mixtures for test agents, the database is useful forlibrary specification/description. The database also providesdocumentation of assay results and the ability to rapidly retrieve,correlate (or conduct other statistical analysis), and evaluate assaydata.

Thus, in some preferred embodiments, the assays of this inventionadditionally involve entering test agent(s) identified as positive(i.e., having an effect on microtubule polymerization, and/ordepolymerization, and/or severing) in a database of “positive” compoundsand more preferably in a database of therapeutic or bioagricultural leadcompounds.

The database can be any medium convenient for recording and retrievinginformation generated by the assays of this invention. Such databasesinclude, but are not limited to manual recordation and indexing systems(e.g., file-card indexing systems). However, the databases are mostuseful when the data therein can be easily and rapidly retrieved andmanipulated (e.g., sorted, classified, analyzed, and/or otherwiseorganized). Thus, in a preferred embodiment, the signature the databasesof this invention are most preferably “automated” (e.g., electronic[e.g., computer-based]) databases. The database can be present on anindividual “stand-alone” computer system, or a component of ordistributed across multiple “nodes” (processors) on a distributedcomputer systems. Computer systems for use in storage and manipulationof databases are well known to those of skill in the art and include,but are not limited to “personal computer systems,” mainframe systems,distributed nodes on an inter- or intra-net, data or databases stored inspecialized hardware (e.g., in microchips), and the like.

VI. Kits for Screening for Modulators of Microtubule Depolymerization orMicrotubule Severing Agents.

In still another embodiment, this invention provides kits for thepractice of any of the methods described herein. The kits comprise oneor more containers containing one or more of the assay componentsdescribed herein. Such components include, but are not limited tostabilized microtubules, microtubule depolymerizing or microtubule.severing proteins or protein subunits, one or more test agents, reactionmedia, solid supports (e.g., microtitre plates) with attachedcomponents, buffers, labels, and other reagents as described herein.

The kits may optionally include instructional materials containingdirections (i.e., protocols) for carrying out any of the assaysdescribed herein. While the instructional materials typically comprisewritten or printed materials they are not limited to such. Any mediumcapable of storing such instructions and communicating them to an enduser is contemplated by this invention. Such media include, but are notlimited to electronic storage media (e.g., magnetic discs, tapes,cartridges, chips), optical!media (e.g., CD ROM), and the like. Suchmedia may include addresses to internet sites that provide suchinstructional materials.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1

Katanin, a Microtubule-Severing Protein is a Novel AAA ATPase thatTargets to the Centrosome using a WD40-Containing Subunit.

RESULTS

To begin dissecting the functional domains of katanin, we isolated cDNAclones for the p60 and p80 subunits from cDNA derived from sea urchin(Strongylocentrotus purpuratus) egg MRNA. After first obtaining peptidesequence of several proteolytic fragments from the two sea urchinkatanin subunits, cDNA clones were isolated using a combination ofdegenerate PCR, cDNA library screening, and anchor-ligated PCR (seeExperimental Procedures). The predicted amino acid sequences of the cDNAclones contained 139 amino acids (a.a.) and 306 amino acids of peptidesequences obtained by direct microsequencing of p60 and p80respectively.

p60 is Novel Member of the AAA ATPase Superfamily

Sequence analysis of the p60 cDNA clone revealed an open reading framethat encodes a 516 a.a. polypeptide (FIG. 1A). A BLAST search with thepredicted p60 protein sequence revealed that this polypeptide contains aC-terminal domain (a.a. 231-447) that is highly conserved in the AAAATPase superfamily (FIG. 1B) (Confalonieri et al. (1995) BioEssays 17:639-650). This ˜220 amino acid region contains the “Walker A” (P-loop)and “Walker B” motifs found in many ATPases (Walker et al. (1982) EMBOJ. 1: 945-95 1). AAA proteins, which contain either one or two of these220 a.a. ATP-binding modules, constitute a large superfamily whosemembers have been implicated in a variety of cellular functions(Confalonieri et al. (1995) supra.).

Of the AAA domains entered into sequence data bases, mei-1, a C. elegansprotein required for meiosis (Clark-Maguire et al. (1994) Genetics 13 6:533-546), is most closely related to p60 (55% a.a. identify, FIG. 1B).Mei-1 was discovered in a genetic screen as a protein that is requiredfor meiotic spindle formation, but disappears during subsequent mitoticdivisions. Interestingly, both p60 (McNally et al. (1996) J Cell Sci.109: 561-567) and mei- I (Clark-Maguire et al. (1994) J. Cell Biol. 126:199-209) are localized to spindle poles in a microtubule-dependentmanner. However, the N-terminal half of p60 has no significant homologyto mei-1, suggesting that p60 and mei-1 may not be orthologs. BLASTsearches with p60 sequences, however, revealed several human ESTs(expressed sequence tags) that have strong amino acid identity outsideof the AAA domain, suggesting the existence of vertebrate homologs ofp60.

p80 Contains WD40 Repeats

Sequence analysis of the sea urchin p80 cDNA clone revealed a predicted690 a.a. polypeptide that contains six “WD40” repeat motifs extendingfrom residues 1-256 (FIG. 2A). An alignment of these repeats with twounrelated WD40 repeat-containing proteins is shown in FIG. 2B. The WD40repeats in several proteins have been documented to participate inprotein-protein binding interactions (Komachi et al. (1994) Genes Dev.8: 2857-2867; Wall et al. (1995) Cell 83: 1047-1058). The C-terminalregion of p80 (residues 257-690) did not exhibit significant amino acididentity to any previously described protein. However, significantidentity of sea urchin p80 was observed with several human EST clones.The sequences of these clones were used to isolate a full length humanp80 katanin homolog by PCR (see Experimental Procedures). The human cDNAencodes a predicted 655 a.a. protein with 61% a.a. identity in the WD40domain (a.a. 1-256) (FIG. 2B), 23% a.a. identity in the central 187residues, and 54% a.a. identity in the C-terminal 164 a.a. with S.purpuratus p80 katanin (latter two regions are not shown).

Baculovirus Expression and Molecular Structure of the Katanin Subunits

Deciphering the roles of the two katanin subunits is essential forunderstanding the enzyme's mechanism and biological activities. However,separation of the native sea urchin p60/p80 subunits requires denaturingconditions. We therefore sought to express the two subunits together andseparately and then test their enzymatic activities. Bacterialexpression of p60 produced largely insoluble protein, and the smallamount of soluble p60 had no microtubule-stimulated ATPase activity(data not shown). However, using the baculovirus expression system, weobtained soluble p60, p80, and the p60/p80 complex (each expressed witha N-terminal His₍₆₎ tag (SEQ ID NO:14), and purified the expressedproteins using metal affinity chromatography (FIG. 3A). When p60 and p80were co-expressed, the stoichiometry of the two subunits in the purifiedprotein was approximately equal (1.0:0.9 p60:p80 molar ratio, asdetermined by Coomassie staining). Moreover, immunoprecipitation with ananti-p60 antibody led to co-immunoprecipitation of equal quantities ofp60 and p80 (FIG. 3B). These results indicate that baculovirus-expressed p60 and p80 heterodimerize, as observed with native katanin(McNally and Vale (1993) Cell, 75: 419-429).

To examine katanin's structure, baculovirus-expressed p60, p80, orp60/80 was adsorbed onto mica chips, and the chips were subsequentlyfrozen, etched, and rotary shadowed with platinum (Heuser (1989) J.Electron Microsc. Technique 13:, 244-263; Heuser (1983) J. Mol. Biol.169: 155-195). The platinum-shadowed p60 appeared as a 14-16 nm ringpunctuated in the center by a 3-5 nm opening, often with what appears tobe cracks radiating outward (FIG. 4A). p80, on the other hand, appearedas ˜11 nm particles and occasional unstructured protein aggregates;rings were not observed (FIG. 4B). Rings were also seen for p60/p80complexes (FIG. 4C, 4D) and native sea urchin katanin (data not shown).Interestingly, two types of p60/p80 complexes were visible: large ˜20 nmdiameter rings with bright edges, which is suggestive of tallercomplexes that extend upward from the mica (FIG. 4D), and smaller ringsof the size of p60 alone with several p80 sized particles radiating fromthe central ring (FIG. 4C). The large and small rings might representclosed and “splayed” versions of the p60/p80 complex, respectively,which could be produced if the complex dissociates upon mica adsorption.Both p60 and p60/p80 structures resemble the rings observed for the AAAATPases NSF and p97, whose dimensions are 15-17 nm (Hanson et al. (1997)Cell 90: 523-535).

p60 Katanin has Microtubule-Stimulated ATPase and Severing Activity

With the availability of isolated p60 and p80, we then examined whetherthe individual subunits have ATPase activity. The co-expressed p60/p80heterodimer displayed an ATP turnover rate of 0.3 ATP/sec/heterodimer;this activity was stimulated ˜10-fold by microtubules (FIG. 5A). Thisbasal activity and the fold stimulation by microtubules are similar tothat observed for native sea urchin katanin (data not shown). Consistentwith the finding of an AAA domain in its sequence, p60 alone displayed amicrotubule-stimulated ATPase activity. Surprisingly, the maximal basaland microtubule-stimulated ATPase rates of p60 were only 2-fold lowerthan those of the p60/p80 heterodimer (FIG. 5A). p80 itself had nodetectable ATPase activity. The activation of ATPase activity bymicrotubules displayed an atypical, non-hyperbolic behavior. ATPturnover by p60 and p60/p80 was stimulated at low concentrations ofmicrotubules (peak at ˜2 μM tubulin), but then decreased at highermicrotubule concentrations (FIG. 5A). This same complex pattern ofmicrotubule stimulation was also observed for native sea urchin katanin(data not shown).

We then tested the microtubule severing activity of p60, p80, andp60/p80 using a fluorescence microscopy assay (McNally and Vale (1993)Cell, 75: 419-429). Both p60 and p60/p80 severed microtubules in thisassay (FIG. 5B). Broken microtubules were observed within 1 min afterintroducing 0.1 μM p60 or p60/p80, and microtubules were completelydisassembled after 5 min. The reaction appeared somewhat slower with p60alone. Microtubules remained intact if ATP was omitted from the reaction(not shown). In contrast, p80 was unable to sever microtubules atconcentrations 5-fold higher than those used for p60 (FIG. 5B). Theseexperiments demonstrate that p60 alone can carry out all of the stepsnecessary for coupling ATP hydrolysis to microtubule disassembly.

To better compare the microtubule severing activities of p60 andp60/p80, we developed a quantitative microtubule disassembly assay basedupon a previous finding that DAPI fluorescence intensity is higher whenthis dye is bound to polymerized versus free tubulin (Heusele et al.(1987) Eur. J. Biochem. 165: 613-620). When katanin and ATP wereincubated with DAPI-labeled microtubules, a linear decrease influorescence intensity was observed as a function of time, reflectingthe conversion of microtubules to tubulin (FIG. 5C. The loss ofmicrotubule polymer was confirmed by centrifugation studies, whichshowed an increase in non-sedimentable tubulin with a similar timecourse (data not shown). The fluorescence decrease induced by theseenzymes reached a steady-state level that was slightly higher than pure,monomeric tubulin, suggesting that some tubulin oligomer may exist atsteady state. The rate of fluorescence decrease was proportional to p60or p60/p80 concentration over a 10-fold range (data not shown). When therates of microtubule disassembly were compared, p60 was half as activeas p60/p80 (FIG. 5C). This slower rate of microtubule disassembly isconsistent with the previously described 2-fold decrease in ATPaseactivity of p60 compared with p60/p80.

The p80 WD40 Domain Targets to the Centrosome

The finding that p60 by itself can sever microtubules left open thequestion of the function of the p80 katanin subunit. At least twofunctional domains of p80 could be postulated. First, since katanin is aheterodimer (McNally and Vale (1993) Cell, 75: 419-429), some part ofp80 must be involved in heterodimerization with p60. Second, becauseprevious studies have shown that katanin is concentrated at centrosomesin vivo (McNally et al. (1996) J. Cell Sci. 109: 561-567), p80 couldcontain a domain that interacts with a centrosomal protein to allowtargeting of the katanin holoenzyme. Because WD40 repeats have beenimplicated in heterophilic protein-protein interactions (Komachi et al.(1994) Genes Dev. 8: 2857-2867; and Wall et al. (1995) Cell 833:10471058), the six WD40 repeats in p80 represented a good candidatedomain for participating in either dimerization or centrosome targeting.

In order to test whether the WD40 repeats of p80 are required forheterodimerization with p60, we deleted the entire WD40 domain andexamined whether the truncated p80 (p80Δ1-302) interacted with p60 whenthe two polypeptides were co-translated in a rabbit reticulocyte system.The truncated p80 (p8Δ1-302) was co-immunoprecipitated by the anti-p60antibody only in the presence of p60 and just as efficiently as fulllength p80 (FIG. 6). This finding indicates that the WD40 repeats arenot required for dimerizing the two katanin subunits. Nevertheless, itremained possible that the WD40 domain was one of multiple, redundantp60-interacting domains. However, a C-terminal truncation of p80 (p80 Δ303-690) containing only the WD40 domain did not coimmunoprecipitate withp60 (FIG. 6). These results indicate that the p80 WD40 repeats areneither necessary nor sufficient for dimerization with p60. To determinewhich region of p80 is required for interaction with p60, a p80 deletionlacking the C-terminal 130 amino acids (p80Δ560-690) was constructed andwas found not to co-inmmunoprecipitate with p60 (FIG. 6). These findingssuggest that the C-terminal 130 amino acids of p80, but not the WD40repeat domain, are involved in the dimerization with p60.

To examine whether the p80 WD40 repeats bind to a protein in thecentrosome, we tested whether these repeats can target a heterologousprotein (green fluorescent protein, GFP) to the centrosome aftertransient transfection in the human fibroblast cell line, MSU1.1 (Lin etal. (1995) Int. J. Cancer 63: 140-147). The WD40 domain of human p80katanin was used, because it was more likely that the human proteinwould interact with centrosomal proteins in this human cell line.Immunofluorescence of MSU1.1 cells with an antibody specific for humanp80 katanin (see Experimental Procedures) showed labeling of thecytoplasm and more concentrated staining at one or two spots thatco-localized with γ-tubulin staining (FIG. 7), confirming thatendogenous katanin is concentrated at centrosomes in fibroblasts as- itis in sea urchin embryos (McNally et al. (1996) J. Cell Sci. 109:561-567). In contrast to the localization in sea urchin embryos, theconcentration of p80 at centrosomes in fibroblasts remained aftercomplete depolymerization of microtubules with nocodazole (data notshown), suggesting that katanin is bound to the pericentriolar material.When a fusion protein consisting of the six WD40 repeats of human p80katanin (a.a. 1-263) appended to the N-terminus of green fluorescentprotein (GFP) was expressed in MSU1.1 cells, one or two foci of greenfluorescence that co-localized with γ-tubulin staining was observed 2-4hr after transfection in addition to diffuse cytoplasmic fluorescence(FIG. 7). Identical results were obtained in transfections of HeLa cells(not shown). In contrast to these findings, cells transfected with GFPalone never revealed foci of green fluorescence at centrosomes (notshown). After longer periods of expression (8-24 hr) of p80 WD40-GFP,numerous heterogeneously-sized bright foci of green fluorescenceappeared that did not co-localize with γ-tubulin, and later, massiveaggregates several μm in diameter were observed (not shown). Theseresults indicate that the WD40 repeats of human p80 katanin aresufficient to target GFP to the centrosome and suggest that once thecentrosome binding sites are saturated, the additional fusion proteinaggregates in the cytoplasm.

Discussion

Katanin is a unique enzyme that couples ATP hydrolysis to thedissociation of tubulin subunits from the microtubule lattice (McNallyand Vale (1993) Cell, 75: 419-429). Other than the motor proteinskinesin and dynein, katanin is the only known microtubule-associatedATPase. In this study, we have determined the primary structure of thep60 and p80 katanin subunits and examined the roles of the two subunitsin microtubule severing and the cellular localization of the enzyme.

Mechanism of Katanin-Mediated Microtubule Severing

Sequence analysis of p60 katanin revealed that it is a novel member ofthe AAA family of ATPases. This finding suggested that p60 might beresponsible for the previously reported ATPase activity of the nativekatanin dimer (McNally and Vale (1993) Cell, 75: 419-429). However,neither p60 nor p80 contained an identifiable microtubule bindingsequence, such as those found in tau (Butner and Kirschner (1991) J.Cell Biol., 115: 717-730) or MA.PIB (Noble et al. (1989) J. Cell Biol.109: 3367-3376), and therefore it was not possible to ascribe themicrotubule binding and severing activities of katanin to either subunitbased upon sequence information alone. By measuring the activities ofthe p60 and p80 subunits purified individually and together as a dimer,we have found that katanin's p60 subunit exhibits bothmicrotubule-stimulated ATPase activity and microtubule-severing activityin the absence of the p80 subunit. Since p60 has all elements requiredfor functional interactions with microtubules, future structure-functionstudies on the mechanism of microtubule severing can be focused on thissingle subunit. Furthermore, we have found that p60 katanin can formrings, the dimensions and appearance of which are similar to thosereported for the AAA proteins NSF and p97 (Hanson et al. (1997) Cell 90:523-535). The comparison of p60 with other AAA proteins provides cluesas to how katanin disassembles microtubules, as discussed below.

The ATPase properties of katanin show both similarities and differenceswith other AAA family members. Katanin's basal ATPase activity of 0.3ATP/katanin/sec and maximal microtubule-stimulated rate of 3ATP/katanin/sec are comparable to values of 1 ATP/sec for p97 (Peters etal. (1992) J. Mol. Biol., 223: 557-571) and 0.08 ATP/sec for recombinantNSF (Morgan et al. (1994) J. Biol. Chem. 269: 29347-29350). NSF ATPaseis also stimulated two-fold upon binding to its target protein, α- orγ-SNAP (Morgan et al. (1994) J. Biol. Chem. 269: 29347-293 50). However,katanin's ATPase activity displays a complex stimulation bymicrotubules. At low microtubule concentrations (<2 μtM), ATPaseactivity increases with increasing microtubule concentration, but athigher microtubule concentrations, ATPase activity decreases until iteventually approaches basal levels. In contrast, stimulation of kinesinATPase by microtubules (Gilbert et al. (1993) Biochemistry 32:4677-4684) displays typical hyperbolic curves that reach saturation.

At least two potential explanations could account for the unusual ATPasebehavior of katanin. One possibility is that katanin binds microtubulesat two sites, which could elevate the local microtubule concentration bycrosslinking and thereby stimulate katanin's ATPase activity. At highermicrotubule concentrations, however, the ratio of katanin tomicrotubules would be lower, resulting in a less-crosslinked network andless stimulation of ATPase activity. In support of this idea, bundlingof microtubules by katanin has been observed by microscopy (unpublishedobservations). This behavior has been seen in anothercytoskeletal-polymer stimulated ATPase, Acanthamoeba myosin 1, which hastwo discrete actin binding sites: a low affinity catalytic site and ahigher affinity site not involved in catalysis (Lynch et al. (1986) J.Biol. Chem. 261: 17156-17162).

A second explanation for katanin's complex enzymatic behavior couldinvolve katanin oligomerization into rings. Rotary-shadowing EM imagesshow oligomeric ring structures in katanin preparations; howeverhydrodynamic experiments with both native (McNally and Vale (1993) Cell,75: 419-429) and recombinant katanin (data not shown) suggest that themajority of the protein is monomeric. One hypothesis is thatmicrotubules promote p60-p60 oligomerization, and that the assembly ofp60 monomers into a higher order structure on the microtubule stimulatesATPase activity. According to this idea, low microtubule concentrationswould facilitate multimerization, since p60 monomers would be morelikely to bind near one another on the microtubule. High microtubuleconcentrations, on the other hand, would inhibit p60 assembly bysequestering p60 monomers at noncontiguous sites on the lattice. Selfassembly into rings also has been suggested as the cause of dynamin'sbiphasic stimulation of GTPase activity (Tuma and Collins (1994) J.Biol. Chem. 269: 30842-30847; and Warnock et al. (1996) J. Biol. Chem.271: 22310-22314). Cryo-electron microscopy studies of thep60-microtubule complex provide a means of testing this hypothesis.

Based upon studies of other AAA family members, katanin oligomers/ringsmay prove to be important in the severing mechanism. Although servingdiverse functions, many AAA proteins appear to share a common fuinctionas nucleotide-dependent molecular chaperones that disassemble proteincomplexes (Confalonieri and Duguet (1995) supra.). The best studied AAAmember is NSF, which binds to and induces the disassembly of ternarySNARE complexes after hydrolysis of ATP (Hanson et al. (1995) J. Biol.Chem. 270: 1695 5-16961; and Hayashi et al. (1995) EMBO J. 14:2317-2325). This reaction plays a role either in vesicle fusion and/orrecycling of components in membrane trafficking pathways. Recently,electron microscopy studies have revealed that the NSF ring structureadopts extended and compact conformations in the ATP-γ-S and ADP states,respectively (Hanson et al, (1997) Cell 90: 523-535). If attached atseveral points to a protein complex, this transition could break apartbonds in the SNARE complex (Hanson et al., (1997) supra.). Katanin maywork in an analogous fashion. A ring of katanin's dimensions couldpotentially contact multiple tubulin sites on a microtubule, and astructural change during ATP hydrolysis could shift the positions oftubulin binding sites with respect to one another, which would disruptthe microtubule lattice. Another possibility is that katanin acts morelike an ATP-regulated version of actin severing proteins, which arethought to compete for sites at protein-protein interfaces within thepolymer. In this type of mechanism, the AAA domain could serve as anATP-dependent protein clamp that binds tightly to and disruptstubulin-tubulin interfaces during particular steps in the ATPase cycle.

Targeting of Katanin to Centrosomes

Our studies show that p80 does not constitute an essential element ofkatanin's enzymatic mechanism. The finding that p80 is not required formicrotubule-severing activity was somewhat surprising, because all ofthe p60 immunoprecipitates with p80 from sea urchin cytosol (unpublishedobservations). However, experiments reported here have uncovered a rolefor p80 in targeting katanin to centrosomes in vivo. This conclusion isbased upon the finding that the WD40 domain of p80 can target GFP to thecentrosome in cultured human cell lines. Because the WD40 domain cannotdimerize with endogenous p60, the centrosomal localization must be dueto direct interaction of the WD40 domain with one or more residentcentrosomal proteins. WD40 domains are thought to form a conserved betapropeller structure, as first determined for the beta subunits oftransducin and G_(i) (Wall et al. (1995) Cell 83: 1047-1058; Sondek etal. (1996) Nature 379: 369-374). However, each WD40 domain exhibits veryspecific heterophilic protein interactions; exposed residues in the betasubunit of G_(i) interact with the alpha subunit (Wall et al. (1995)supra.), whereas the corresponding residues in the WD40 transcriptionfactor TUP1 mediate binding to a second transcription factor α2(Komachi, and Johnson (1997) Mol. Cell Biol. 17: 6023-6028). Since the Gprotein beta subunits interact with multiple partner proteins (Wall etal. (1995) Cell 83: 1047-1058; and Gaudet et al. (1996) Cell, 87:577-588), it is also possible that the p80 katanin WD40 domain caninteract with more than one protein in vivo. p80 is the only knowncentrosomal protein with a WD40 motif. The findings that katanin has anentire subunit devoted to centrosome localization and that this subunitis conserved between mammals and echinoderms suggest an important rolefor katanin at the centrosome.

The WD40 domain of p80 katanin represents the first example of astructural motif that targets a protein to the centrosome in mammals,although a centrosome-targeting domain has been defined for theDrosophila protein CP190 (Oegema et al. (1995) J. Cell Biol. 131:1261-1273). This provides an opportunity to identify the centrosomalcomponent(s) responsible for anchoring katanin. Further information onthe docking of katanin to the centrosome may provide clues regardingkatanin's role in microtubule disassembly at this organelle.

Experimental Procedures

Peptide Microsequencing

Katanin was purified from extracts of S. purpuratus eggs essentially asdescribed previously (McNally and Vale (1993) Cell, 75: 419-429), exceptthat the hydroxyapatite chromatography was carried out using a PharmaciaHRI. 0/3 0 column packed with 20 μm, ceramic hydroxyapatite beads(American International Chemical, Natick, Mass.). Internal peptidesequences of the p60 and p80 subunits were obtained from native seaurchin katanin as described (Iwamatsu (1992) Electrophoresis 13:142-147). Two additional p80 peptides were also obtained: DASMMAM (SEQID NO:15) and IQGLR (SEQ ID NO:16). p60 Cloning

A cDNA encoding a 400 bp fragment of the p60 subunit (corresponding toa.a. 214374) was cloned from S. purpuratus first strand cDNA usingnested PCR with degenerate oligonucleotides. This fragment was then usedto screen a lambda ZAP-Express cDNA library made from S. purpuratusunfertilized egg MRNA by hybridization. Several independent positiveclones were isolated. One clone was completely sequenced (GENBANKaccession #AF052191). p80 Cloning

An initial partial cDNA clone of p80 katanin was obtained by screeningan S. purpuratus unfertilized egg cDNA library (Wright et al. (1991) J.Cell Biol. 113: 817-833) with an antibody specific for p80 katanin,anti-p81^(aff) (McNally et al. (1996) J. Cell Sci. 109: 561-567). Theinsert of the initial clone was used to isolate a longer cDNA clone(pFM18) from the same library by plaque hybridization. A cDNA cloneencoding the 5′ end of p80 katanin (pFM23) was obtained byanchor-ligated PCR (Apte and Siebert (1993) Biotechniques 15: 890-893)using primers derived from pFM18 sequences and reverse transcriptionreactions utilizing S. purpuratus unfertilized egg mRNA as template. Afull-length p80 cDNA (GENBANK accession #AF052433) was generated byjoining the inserts of pFM1 and pFM23 at a common BstXI site.

BLAST searches of GENBANK with p80 sequences revealed homology with ahuman infant brain cDNA (GENBANK accession #T 16102) which was obtainedand sequenced. Sequences obtained from the T 16102 clone were used toobtain multiple 3′end cDNA clones by 3′ RACE from HT1080 (humanfibrosarcoma) total RNA. An overlapping cDNA clone (pFM54) containingthe translation start site was obtained by PCR amplification from anadult human hippocampal cDNA library (Stratagene, Inc.). Sequenceanalysis of partial cDNAs PCR amplified from HT1080 total RNA or fromthe hippocampal library were over 98% identical in predicted a.a.sequence. The complete DNA sequence of human p80 katanin is availablefrom GENBANK (accession #AF052432).

Antibody Production and Immunoprecipitation

The full-length S. purpuratus p60 cDNA coding sequence was inserted intopMA.LC2 (New England Biolabs) and expressed as a C-terminal fusion tomaltose binding protein in E. coli. Soluble MBP-p60 fusion protein waspurified on an amylose affinity column, eluted with maltose, andinjected into rabbits (antiserum production by BABCO, Berkeley, Calif.).To select p60-specific antibodies that do not react with other AAAmembers, antibodies recognizing the N-terminal non-AAA domain of p60were affinity purified on an Affi-Gel column coupled with the N-terminalresidues 1- 152 of p60 fused to MBP (Harlow and Lane (1988). Antibodies:A Laboratory Manual. (Cold Spring Harbor, N.Y.: Cold Spring HarborLaboratory Press)). The resulting affinity-purified antibody recognizeda single 60 kDa polypeptide in immunoblots of S. purpuratus unfertilizedegg extract.

To prepare a specific antibody to human p80 katanin, the full lengthhuman p80 cDNA was ligated into the E. coli expression vector, pET-28a⁺(Novagen) as a BamHI-XhoI fragment. The protein was expressed and thenpurified in a denatured state in 8 M urea. by nickel chelatechromatography on His-Bind Resin (Novagen). Rabbits were immunized withpolyacrylamide slices containing SDS-PAGE resolved human p80 katanin.Resulting serum was affinity purified with CNBr Sepharose-coupled,bacterially-expressed human p80 katanin. The resulting affinity purifiedantibody recognized a single 80 kDa polypeptide in immunoblots of SDSsolubilized HeLa cells (not shown).

For immunoprecipitations used to demonstrate association ofbaculovirus-expressed S. purpuratus p60 and p80, affinity purifiedanti-p60 antibodies were covalently crosslinked to protein A Sepharoseusing 20 mM dimethylpimilidate (Harlow and Lane (1988) supra.). Afterequilibration in TBST, 20-40 μl of antibody beads were added to kataninsamples diluted in TBST containing 1-2 mg/ml soybean trypsin inhibitor(SBTI). The immunoprecipitations were incubated at 4° C. for 1-2 hr,washed five times with 1 ml of ice-cold TBST, and eluted inSDS-containing sample buffer.

Baculovirus Expression and Purification of Katanin

Katanin subunits were expressed using the Bac-to-Bac™ baculovirusexpression system (Life Technologies), a commercial version of thesite-specific transposition system for making recombinant baculovirus(Luckow et al. (1993) J. Virology 67: 4566-4579). p60 and p80 cDNAcoding sequences were each PCR amplified (Expand polymerase, BoehringerMannheim) and then subcloned separately into pFastBac HT, which resultedin the fusion of a 6xHis Ni2⁺ binding sequence to the N-terminus of bothp80 and p60. A p60-p80 coexpression virus was made by cloning thecomplete p60-FastBac HT and p80-FastBac HT coding regions into thetransfer vector, pDual. Recombinant baculoviruses were preparedaccording to the Life Technologies protocol.

Sf9 cells were grown in SFM-900 II SFM (Life Technologies) supplementedwith 100× antibiotic/antimycotic (Life Technologies) to 0.5× using theshaker culture method (Weiss et al. (1995) pp. 79-85 in BaculovirusExpression Protocols, C. D. Richardson, ed. Totowa, N.J.: Humana PressInc.). Expression of katanin subunits was performed in 11 flaskscontaining 200-300 ml of media using a multiplicity of infection of0.5-1.0 pfu/cell. The cells were harvested at approximately 72 hr postinfection by low speed centrifugation and resuspended in lysis buffer(50 mM Tris pH 8.5, 300 mM NaCl, 2 mM MgCl₂, 20 mM imidazole, 10 mM2-mercaptoethanol, 1 mM ATP, 1 μg/ml pepstatin, 1 μg/ml leupeptin, 1μg/ml aprotinin) before freezing in liquid nitrogen, and storage at −80°C.

To purify the expressed subunits, frozen cells were thawed and DNA wassheared by two passes through a Bio-Neb Cell Disrupter (100 psi helium,13 1/min). Cell debris was removed by centrifugation (40,000×g for 45min). Subunits were bound in batch to Ni²⁺-NTA Superflow (QIAGEN),washed [20 mM Tris pH 8.0, 1 M NaCl, 2 mM MgCl₂, 40 mM imidazole, 0.02%Triton X- 100, 10 mM 2-mercaptoethanol, 0.5 mM ATP] and eluted [20 mMTris pH 8.0, 100 mM NaCl, 150 mM imidazole, 2 mM MgCl₂, 0.02% Triton X-100, 10 mM 2-mercaptoethanol, 100 μM ATP], followed by freezing inliquid nitrogen. Additional purification was sometimes performed byanion-exchange chromatography. Katanin concentrations were estimated bycomparison with BSA standards using either a commercial Bradford reagent(Bio-Rad) or by densitometric analysis of Coomassie-stained SDS-PAGEgels with NIH-IMAGE after image capture on a CCD-based imaging system(Foto/Analyst, Fotodyne).

Electron Microscopic Imaging

Proteins were adsorbed to mica, freeze-dried, and platinum replicatedaccording to established procedures (Heuser (1989) J. Electron Microsc.Technique 13):, 244-263; Heuser (1983) J. Mol. Biol. 169: 155-195).Sample preparation and imaging were similar to that used in the imagingof NSF (Hanson et al. (1997) Cell 90: 523-535), except that mica flakeswere washed with a buffer consisting of 10 mM K-HEPES (pH 7.5), 2 mMMgCl₂, 1 mM nucleotide (ATP or ATP-γ-S). Images were processed usingAdobe Photoshop and displayed at 300,000×.

ATPase Assays

ATPase activity was measured by a modified malachite green method(Kodama et al. (1986) J. Biochem. 99: 1465-1472). ATPase reactions of50-100 μl, were carried out in a buffer previously used for measuringthe ATPase activity of native katanin [20 mM K-HEPES pH 8.0, 25 mMpotassium glutamate 2 mM MgCl₂, 10% glycerol (v/v), 0.02% Triton X-100(w/v), 1 mg/ml BSA] (McNally and Vale (1993) Cell, 75: 419-429), exceptthat soybean trypsin inhibitor (SBTI) was replaced by BSA as a carrierprotein because SBTI increased background phosphate contamination. AnATP regenerating system, consisting of 0.5-1.0 mM phosphoenol pyruvateand 2 units of pyruvate kinase, was included to minimize the inhibitionby ADP observed previously for native katanin (McNally and Vale (1993)Cell, 75: 419-429). Microtubules were prepared from bovine brain tubulin(Hyman et al. (1990) Meth. Enzymol. 196: 303-319; and Williams and Lee(1982) Meth Enzymol. 85B: 376-385). After assembly, microtubules weresedimented (230,000×g; 10 min), resuspended in ATPase buffer lackingBSA, and the polymers were resuspended by repeated passage through a 27gauge needle. Microtubule concentration was determined by measuring theabsorbance at 275 nm in 6 M guanidine HCl by using a molecular mass of110 kDa and an extinction coefficient of 1.03 ml mg⁻¹ cm⁻¹ (Hackney(1988) Proc. Natl. Acad Sci. USA, 85: 6314-6318). ATPase reactions werecarried out at room temperature, and were initiated by addition ofkatanin.

Severing Assays

Microscope-based severing assays were performed using previouslypublished procedures (McNally and Vale (1993) Cell, 75: 419-429), exceptthat microtubules were immobilized by first perfusing flow cells with abacterially expressed kinesin mutant that binds strongly to microtubulesbut is unable to hydrolyze ATP (K560, G234A mutant; R. Vale and E.Taylor, unpublished results). Assays were performed in 20 mM Hepes (pH7.5), 2 mM MgCl₂, 1 mM ATP with an oxygen scavenger system consisting ofglucose oxidase (220 μg/ml), catalase (36 μg/ml), glucose (22.5 mM), and2-mercaptoethanol (71.5 mM). Images were captured using a cooled,slow-scan CCD (Photometerics) and processed using Adobe Photoshop.

DAPI severing assays were performed using conditions where the change influorescence intensity was linear with the amount of tubulin polymeradded (Heusele et al. (1987) Eur. J. Biochem. 165: 613-620). Severingreactions containing 2 μM microtubules (polymerized and resuspended inATPase buffer as above) were incubated with 10 μM DAPI, along with 1 mMATP, 10 mM phosphoenol pyruvate, 250 μg/ml pyruvate kinase (BoehringerMannheim), and 1 mg/ml BSA. The reaction volume was 80 μL, andfluorescence intensity was measured by exciting at 370 nm. and measuringthe emission at 450 mn using a model 8100 fluorimeter (SLM Instruments)in photon counting mode.

In vitro Translation Co-Immunoprecipitation

In order to facilitate the non-radioactive detection of in vitrotranslated p60 and p80, each cDNA was ligated into the vector pCITE-4a+(Novagen) such that the proteins would be translated in frame with a 37amino acid N-terminal S-Tag. In vitro synthesis of proteins directlyfrom plasmid DNAs was accomplished using the Single Tube Protein System2, T7 (Novagen). For co-immunoprecipitation assays, p60 and p80constructs were usually co-expressed. However, identical results wereobtained if the constructs were expressed separately and the incubatedtogether for 30 min at room temperature. For immunoprecipitations,lysates were incubated on ice with Pansorbin (Calbiochem)-antibodycomplexes, washed in NET buffer (50 mM Tris-Cl (pH 7.5), 150 mM NaCl,0.1% Nonidet P40, 1 mM EDTA (pH 8.0), 0.25% gelatin and 0.02% sodiumazide), then resuspended in SDS-PAGE sample buffer. In vitro translationproducts in both the pellets and supernatants from theimmunoprecipitations were resolved by SDS-PAGE, transferred tonitrocellulose, probed with S-protein HRP conjugate (Novagen) anddetected by chemiluminescence.

Cell Culture and Transfections and Immunofluorescence

To allow transient expression of a human p80 WD40-GFP fusion protein inHeLa cells, a DNA fragment containing amino acids 1-263 of human p80katanin was generated by PCR amplification, placing a BamHI site and aKozak consensus at the predicted translation start and an EcoRI siteafter the codon for a.a. 263. This BamHI-EcoRI fragment was ligated intothe GFP fusion vector pEGFP-N1 (Clontech).

Both MSU1.1 and HeLa cells were grown on 18 mm glass coverslips inOptimem medium (Life Technologies) supplemented with 10% fetal bovineserum, penicillin and streptomycin. Plasmids were transfected usingSuperfect Reagent (Qiagen) for 2 hr after which coverslips were washedwith PBS and placed in fresh culture medium at 37° C. with 5% CO₂ for1-24 hr.

For imaging of GFP-fluorescence and immunofluorescence with the humanp80 katanin antibody or with the γ-tubulin antibody, monoclonal GTU88(Sigma Chemical), cells on coverslips were fixed either in −20° C.methanol or in 0.5× PBS, 3.7% formaldehyde, 75% methanol at 22° C. for10 min followed by rehydration in TBST. Antibody labelling was carriedout in TBST containing 4% BSA. Images were captured with a NikonMicrophot SA microscope, 100× Plan Fluor 1.3 objective, PhotometricsQuantix camera and IP Lab Spectrum software (Scanalytics).

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1-25. (canceled)
 26. An isolated polypeptide having microtubule severingactivity, said polypeptide comprising an amino acid sequence that isencoded by a nucleic acid sequence that hybridizes under stringentconditions with a nucleic acid sequence that encodes SEQ ID NO:1. 27.The polypeptide of claim 26, wherein said polypeptide comprises SEQ IDNO:1.
 28. The polypeptide of claim 26, wherein said polypeptide: 1)comprises at least 8 contiguous amino acids of SEQ ID NO: 1; 2) elicitsthe production of an antibody that specifically binds to SEQ ID NO:1;and 3) does not bind to antiserum that is raised against SEQ ID NO:1,and that has been fully immunosorbed with SEQ ID NO:1.
 29. Thepolypeptide of claim 26, wherein said polypeptide is the pelypeptideconsists of SEQ ID NO:1. 30-33. (canceled)
 34. A kit for screening foragents that modulate microtubule depolymerization, said kit comprisingone or more containers containing one or more of an isolated microtubulesevering protein and a microtubule depolymerizing protein.
 35. The kitof claim 34, further comprising a polymerized microtubule labeled with4′-6-diamidino-2-phenylindole (DAPI).
 36. The kit of claim 34, whereinsaid microtubule is stabilized by contact with an agent chosen from oneor more of paclitaxel, paclitaxel analogue, and non-hydrolyzablenucleotide GTP analogue.
 37. The kit of claim 36, wherein saidmicrotubule is attached to a solid surface.
 38. The kit of claim 37,wherein said microtubule is attached to said surface by binding with amotor protein.
 39. The kit of claim 34, wherein said microtubulesevering protein or microtubule depolymerizing protein is selected fromthe group consisting of katanin polypeptide, p60 subunit of kataninpolypeptide, Xenopus kinesin central motor 1 (XKCM1) polypeptide, and astathmin (OP18) polypeptide. 40-41. (canceled)
 42. The kit of claim 34,wherein said one or more of microtubule severing protein and microtubuledepolymerizing protein is attached to a solid surface. 43-59. (canceled)60. The polypeptide of claim 26, wherein said nucleic acid sequence thatencodes SEQ ID NO:1 is listed in Genbank AF052191.
 61. The polypeptideof claim 26, wherein said polypeptide comprises SEQ ID NO:1 havingconservative substitutions.
 62. The polypeptide of claim 26, whereinsaid polypeptide consists of SEQ ID NO:1 having conservativesubstitutions.
 63. The polypeptide of claim 26, wherein saidhybridization conditions comprise hybridization at 42° C. overnight in50% formamide.
 64. The polypeptide of claim 26, wherein said isolatedpolypeptide is recombinant.
 65. An isolated polypeptide comprising SEQID NO:1 and having microtubule severing activity.
 66. An isolatedpolypeptide consisting of SEQ ID NO:1.
 67. The kit of claim 34, furthercomprising tubulin that is labeled with a label chosen from one or moreof 4′-6-diamidino-2-phenylindole (DAPI), anilinonapthalene sulfonate(ANS), bis-ANS (Bis-anilinonapthalene sulfonate), N-phenyl-1-naphthylene(NPN), ruthernium red, cresol violet, and 4-(dicyanovinyl)julolidine(DCVJ).
 68. The kit of claim 34, further comprising tubulin that islabeled with 4′-6-diamidino-2-phenylindole (DAPI).
 69. The kit of claim34, wherein said microtubule depolymerizing protein comprises Xenopuskinesin central motor 1 (XKCM1) polypeptide.
 70. The kit of claim 34,wherein said microtubule depolymerizing protein comprises stathmin(OP18) polypeptide.
 71. The kit of claim 34, wherein said microtubuledepolymerizing protein comprises katanin polypeptide.
 72. The kit ofclaim 34, wherein said microtubule depolymerizing protein comprises ap60 subunit of katanin polypeptide.
 73. The kit of claim 37, whereinsaid microtubule is attached to said surface by binding with a moleculechosen from one or more of inactivated microtubule motor protein,avidin-biotin linkage, anti-tubulin antibody, microtubule bindingprotein (MAP), polyarginine, polyhistidine, and polylysine.
 74. A kitcomprising the polypeptide of claim 26.